Anti-CD40 antibodies

ABSTRACT

The present invention includes compositions and methods for the expression, secretion and use of novel compositions for use as, e.g., vaccines and antigen delivery vectors, to delivery antigens to antigen presenting cells. In one embodiment, the vector is an anti-CD40 antibody, or fragments thereof, and one or more antigenic peptides linked to the anti-CD40 antibody or fragments thereof, including humanized antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/159,059, filed Mar. 10, 2009; U.S. Provisional Application Ser.No. 61/159,055 filed Mar. 10, 2009; and U.S. Provisional ApplicationSer. No. 61/159,062, filed Mar. 10, 2009. The entire contents of eachare incorporated herein by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with U.S. Government support under Contract No.1U19A1057234-0100003 awarded by the NIH. The government has certainrights in this invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of immunization,and more particularly, to novel anti-CD40 antibodies and anti-CD40antibody-based vaccines.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with antigen presentation.

One example of vaccines and methods for antigen presentation is taughtin U.S. Pat. No. 7,118,751, issued to Ledbetter, et al., for DNAvaccines encoding an amino-terminus antigen linked to a carboxy-terminusdomain that binds CD40. Briefly, vaccines are taught that target one ormore antigens to a cell surface receptor to improve the antigen-specifichumoral and cellular immune response. Antigen(s) linked to a domain thatbinds to a cell surface receptor are internalized, carrying antigen(s)into an intracellular compartment where the antigen(s) are digested intopeptides and loaded onto MHC molecules. T cells specific for the peptideantigens are activated, leading to an enhanced immune response. Thevaccine may comprise antigen(s) linked to a domain that binds at leastone receptor or a DNA plasmid encoding antigen(s) linked to a domainthat binds at least one receptor. A preferred embodiment of theinvention targets HIV-1 env antigen to the CD40 receptor, resulting indelivery of antigen to CD40 positive cells, and selective activation ofthe CD40 receptor on cells presenting HIV-1 env antigens to T cells.

Another example is found in United States Patent Application No.20080254026, filed by Li, et al., for antagonist anti-CD40 monoclonalantibodies and methods for their use. Briefly, compositions and methodsare disclosed for use in therapy for treating diseases mediated bystimulation of CD40 signaling on CD40-expressing cells are provided. Themethods comprise administering a therapeutically effective amount of anantagonist anti-CD40 antibody or antigen-binding fragment thereof to apatient in need thereof. The antagonist anti-CD40 antibody orantigen-binding fragment thereof is free of significant agonistactivity, but exhibits antagonist activity when the antibody binds aCD40 antigen on a human CD40-expressing cell. Antagonist activity of theanti-CD40 antibody or antigen-binding fragment thereof beneficiallyinhibits proliferation and/or differentiation of human CD40-expressingcells, such as B cells.

Yet another example is taught in United States Patent Application No.20080241139, filed by Delucia for an adjuvant combination comprising amicrobial TLR agonist, a CD40 or 4-1BB agonist, and optionally anantigen and the use thereof for inducing a synergistic enhancement incellular immunity Briefly, this application is said to teach adjuvantcombinations comprising at least one microbial TLR agonist such as awhole virus, bacterium or yeast or portion thereof such a membrane,spheroplast, cytoplast, or ghost, a CD40 or 4-1BB agonist and optionallyan antigen wherein all 3 moieties may be separate or comprise the samerecombinant microorganism or virus are disclosed. The use of theseimmune adjuvants for treatment of various chronic diseases such ascancers and HIV infection is also provided.

United States Patent Application No. 20080199471, filed by Bernett, etal., is directed to optimized CD40 antibodies and methods of using thesame. Briefly, this application is said to teach antibodies that targetCD40, wherein the antibodies comprise at least one modification relativeto a parent antibody, wherein the modification alters affinity to anFcγR or alters effector function as compared to the parent antibody.Also disclosed are methods of using the antibodies of the invention.

Finally, United States Patent Application No. 20080181915, file byTripp, et al., is directed to a CD40 ligand adjuvant for respiratorysyncytial virus. Briefly, this application is said to teach methods andadjuvants for enhancing an immune response to RSV in a host, wherein themethods and adjuvants comprise a source of a CD40 binding protein.Preferably, the CD40 binding protein is CD40L and the source is a vectorcomprising a promoter operatively linked to a CD40L coding region. Theenhanced immune response produced by the adjuvants and methods of thecurrent invention includes both increased expression of Th1 cytokinesand increased production of antibody.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is a recombinant antibody or anantigen binding fragment thereof, both of which bind to CD40,comprising: at least one antibody light chain variable region of SEQ IDNOS: 2, 4, 5 or 7; and at least one antibody heavy chain variable regionof SEQ ID NOS: 1, 3 or 7. In one aspect, the antibody further comprisesa heavy chain constant region, wherein the heavy chain constant regioncomprises a gamma-1, gamma-2, gamma-3, or gamma-4 human heavy chainconstant region or a variant of the human heavy chain constant region.In one aspect, the antibody further comprises a light chain constantregion, wherein the light chain constant region comprises a lambda or akappa human light chain constant region. In another aspect, the bindingfragment is selected from group consisting of Fab, Fab′, Fab′-SH, Fv,scFv, F(ab′)2, and a diabody. In another aspect, the antibody comprisesthe polypeptide sequence of SEQ ID NOS: 1, 3 or 6, and/or the antibodycomprises the polypeptide sequence of SEQ ID NOS: 2, 4, 5, or 7. Inanother aspect, the antibody is produced by a hybridomaanti-CD40_12E12.3F3 (ATCC Accession No. PTA-9854), anti-CD40_12B4.2C10(Deposit Submission No. HS446, ATCC Accession No. PTA-10653), andanti-CD40_11B6.1C3 (Deposit Submission No. HS440, ATCC Accession No.PTA-10652). In another aspect, the antibody alone is capable of causingdendritic cells to secrete at least one of IL-6, MIP-1a, IL-12p40 orTNFalpha without prior activation of the dendritic cells. In one aspect,the antibody is capable of causing dendritic cells activated with GM-CSFand Interferon alpha to secrete at least one of IL-6, MIP-1a, IP-10,IL-10 or IL-12p40. In another aspect, the recombinant antibody comprisesat least 90, 95, 99 or 100% sequence identity with at least one antibodylight chain variable region of SEQ ID NOS: 2, 4, 5 or 7; and at leastone antibody heavy chain variable region of SEQ ID NOS: 1, 3 or 7. Inanother aspect, the antibody is humanized.

Another embodiment of the present invention is a composition comprisingan antibody or an antigen binding fragment thereof, in combination witha pharmaceutically acceptable carrier or diluent, wherein the antibodyis the antibody of claim 1.

Another embodiment of the present invention is a humanized recombinantantibody or an antigen binding fragment thereof, both of which bind toCD40, comprising: a) at least one antibody light chain variable regionof SEQ ID NOS.: 2, 4, 5 or 7; and b) at least one antibody heavy chainvariable region of SEQ ID NOS.: 1, 3 or 7. In one aspect, the antibodyfurther comprises a heavy chain constant region, wherein the heavy chainconstant region comprises a gamma-1, gamma-2, gamma-3, or gamma-4 humanheavy chain constant region or a variant of the human heavy chainconstant region. In one aspect, the antibody further comprises a lightchain constant region, wherein the light chain constant region comprisesa lambda or a kappa human light chain constant region. In anotheraspect, the binding fragment is selected from group consisting of Fab,Fab′, Fab′-SH, Fv, scFv, F(ab′)2, and a diabody. In another aspect, theantibody, or antigen binding fragment thereof, comprises the polypeptidesequence of SEQ ID NOS.: 1, 3 or 6, and/or the polypeptide sequence ofSEQ ID NOS.: 2, 4, 5, or 7. In one aspect, the antibody comprises atleast the variable region of anti-CD40_12E12.3F3 (ATCC Accession No.PTA-9854), anti-CD40_12B4.2C10 (Deposit Submission No. HS446, ATCCAccession No. PTA-10653), and anti-CD40_11B6.1C3 (Deposit Submission No.HS440, ATCC Accession No. PTA-10652). In another aspect, the humanizedantibody comprises the complementarity determining regions of: a) atleast one antibody light chain variable region of SEQ ID NOS: 2, 4, 5 or7; and b) at least one antibody heavy chain variable region of SEQ IDNOS: 1, 3 or 7 on a human antibody framework.

Another embodiment of the present invention is a composition comprisingan antibody or an antigen binding fragment thereof, in combination witha pharmaceutically acceptable carrier or diluent, wherein the antibodyis the antibody of claim a recombinant antibody or an antigen bindingfragment thereof, both of which bind to CD40, comprising: at least oneantibody light chain variable region of SEQ ID NO.: 2, 4, 5 or 7; and atleast one antibody heavy chain variable region of SEQ ID NO.: 1, 3 or 7.In another aspect, the antibody comprises at least the variable regionof the antibody anti-CD40_12E12.3F3 (ATCC Accession No. PTA-9854),anti-CD40_12B4.2C10 (ATCC Submission No. HS446, Accession No.PTA-10653), and anti-CD40_11B6.1C3 (ATCC Submission No. HS440, AccessionNo. PTA-10652). In another aspect, the antibody comprises at least onevariable domain having 90, 95 99 or 100% sequence identity with a heavychain variable domain of SEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS.: 2,4, 5, or 7.

Another embodiment of the present invention is an isolated nucleic acidencoding the polypeptide of SEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS.:2, 4, 5, or 7. In one aspect, the nucleic acids further comprise nucleicacid sequences from human antibodies that humanize the antibody. Inanother aspect, the antibody comprises at least one variable domainhaving 90, 95 99 or 100% sequence identity with a heavy chain variabledomain of SEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS.: 2, 4, 5, or 7.

Another embodiment of the present invention is an expression vectorcomprising the isolated nucleic acid encoding the polypeptide of SEQ IDNOS: 1, 3 or 6, and/or SEQ ID NOS: 2, 4, 5, or 7, operably linked tocontrol sequences recognized by a host cell transfected with the vector.In another aspect, the antibody comprises at least one variable domainhaving 90, 95 99 or 100% sequence identity with a heavy chain variabledomain of SEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS.: 2, 4, 5, or 7.

Another embodiment of the present invention is a host cell comprisingthe vector that encodes the isolated nucleic acid encoding thepolypeptide of SEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS: 2, 4, 5, or 7.In another aspect, the antibody comprises at least one variable domainhaving 90, 95 99 or 100% sequence identity with a heavy chain variabledomain of SEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS.: 2, 4, 5, or 7.

Another embodiment of the present is a method of producing apolypeptide, comprising culturing the host cell comprising isolatednucleic acid encoding the polypeptide of SEQ ID NOS: 1, 3 or 6, and/orSEQ ID NOS: 2, 4, 5, or 7, under conditions wherein the nucleic acidsequence is expressed, thereby producing the polypeptide, and recoveringthe polypeptide from the host cell. In another aspect, the antibodycomprises at least one variable domain having 90, 95 99 or 100% sequenceidentity with a heavy chain variable domain of SEQ ID NOS: 1, 3 or 6,and/or SEQ ID NOS.: 2, 4, 5, or 7.

Another embodiment of the present invention is an expression vectorcomprising the isolated nucleic acid encoding the polypeptide of SEQ IDNOS: 1, 3 or 6, and/or SEQ ID NOS: 2, 4, 5, or 7, operably linked tocontrol sequences recognized by a host cell transfected with the vector.In another aspect, the antibody comprises at least one variable domainhaving 90, 95 99 or 100% sequence identity with a heavy chain variabledomain of SEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS.: 2, 4, 5, or 7.

Another embodiment of the present invention is a method of producing apolypeptide, comprising culturing the host cell comprising a vector thatcomprises isolated nucleic acid encoding the polypeptide of SEQ ID NOS:1, 3 or 6, and/or SEQ ID NOS: 2, 4, 5, or 7, under conditions whereinthe nucleic acid sequence is expressed, thereby producing thepolypeptide, and recovering the polypeptide from the host cell.

Another embodiment of the present invention is an isolated nucleic acidsequence encoding an antibody specific for CD40 comprising a light chainhaving the nucleic acid sequence of SEQ ID NO: 9, 11, 12 or 15 and aheavy chain having the nucleic acid sequence of SEQ ID NO: 8, 10 or 14.In one aspect, the binding fragment is an antibody fragment selectedfrom the group consisting of Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, anda diabody. In another aspect, the antibody comprises at least onevariable domain having 90, 95 99 or 100% sequence identity with a heavychain variable domain of SEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS.: 2,4, 5, or 7.

Another embodiment of the present invention is a method to identify anacceptor germline sequence for a humanized antibody, which methodcomprises the steps of: a) identifying a non-human antibody that has thedesired biological activity selected from at least one antibody lightchain variable region of SEQ ID NO: 2, 4, 5 or 7; and at least oneantibody heavy chain variable region of SEQ ID NO: 1, 3 or 7; b)determining the amino acid sequence of a non-human antibody VH and VLdomains; and c) comparing the nonhuman antibody sequence to a group ofhuman germline sequences, wherein the comparison comprises the substepsof: 1) assigning the sequence of non-human VH and VL domain sequencesresidue numbers; 2) delineating the CDR and FR regions in the sequence;3) assigning a predetermined numerical score at each residue positionfor which the non-human and human germline sequences are identical; and4) totaling all of the residue scores to generate a total score for eachhuman germline sequence; and d) identifying the human germline sequencewith the highest total residue score as the acceptor germline sequence.In one aspect, the non-human antibody is specific for CD40. In anotheraspect, the antibody comprises at least one variable domain having 90,95 99 or 100% sequence identity with a heavy chain variable domain ofSEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS.: 2, 4, 5, or 7.

Another embodiment of the present invention is an antibody generated bythe method comprising a) identifying a non-human antibody that has thedesired biological activity selected from at least one antibody lightchain variable region of SEQ ID NO: 2, 4, 5 or 7; and at least oneantibody heavy chain variable region of SEQ ID NO: 1, 3 or 7; b)determining the amino acid sequence of a non-human antibody VH and VLdomains; and c) comparing the nonhuman antibody sequence to a group ofhuman germline sequences, wherein the comparison comprises the substepsof: 1) assigning the sequence of non-human VH and VL domain sequencesresidue numbers; 2) delineating the CDR and FR regions in the sequence;3) assigning a predetermined numerical score at each residue positionfor which the non-human and human germline sequences are identical; and4) totaling all of the residue scores to generate a total score for eachhuman germline sequence; and d) identifying the human germline sequencewith the highest total residue score as the acceptor germline sequence.In one aspect, the non-human antibody is specific for CD40. In anotheraspect, the antibody comprises at least one variable domain having 90,95 99 or 100% sequence identity with a heavy chain variable domain ofSEQ ID NOS: 1, 3 or 6, and/or SEQ ID NOS.: 2, 4, 5, or 7.

Another embodiment of the present invention is a method of making anantibody comprising expressing in a host cell a recombinant antibody oran antigen binding fragment thereof, both of which bind to CD40,comprising: at least one antibody light chain variable region of SEQ IDNO: 2, 4, 5 or 7; and at least one antibody heavy chain variable regionof SEQ ID NO: 1, 3 or 7. In one aspect, the host cell is a bacterial,fungal, insect, or mammalian cell. In another aspect, the antibody is ahumanized antibody. In another aspect, the antibody comprises at leastone variable domain having 90, 95 99 or 100% sequence identity with aheavy chain variable domain of SEQ ID NOS: 1, 3 or 6, and/or SEQ IDNOS.: 2, 4, 5, or 7.

Another embodiment of the present invention is a recombinant antibody oran antigen binding fragment thereof that binds to CD40, wherein theantibody alone is capable of causing dendritic cells to secrete at leastone of IL-6, MIP-1a, IL-12p40 or TNFalpha without prior activation ofthe dendritic cells. In one aspect, the antibody comprises at least onevariable domain having 90% sequence identity with at least one antibodylight chain variable region of SEQ ID NOS: 2, 4, 5 or 7; and at leastone variable domain having 90% sequence identity with one antibody heavychain variable region of SEQ ID NOS: 1, 3 or 7. In another aspect, theantibody comprises the polypeptide sequence of SEQ ID NOS: 1, 3 or 6,the polypeptide sequence of SEQ ID NOS: 2, 4, 5, or 7, or both. Inanother aspect, the antibody is produced by a hybridoma selected fromanti-CD40_12E12.3F3 (ATCC Accession No. PTA-9854), anti-CD40_12B4.2C10(ATCC Submission No. HS446, Accession No. PTA-10653), andanti-CD40_11B6.1C3 (ATCC Submission No. HS440, Accession No. PTA-10652).In another aspect, the antibody is humanized. In another aspect, theantibody is capable of causing dendritic cells activated with GM-CSF andInterferon alpha to secrete at least one of IL-6, MIP-1a, IP-10, IL-10or IL-12p40. In another aspect, the antibody the antibody alone iscapable of causing B cell proliferation of at least 10%, 20%, 25%, 28%,30% or 35%.

Another embodiment of the present invention is a recombinant antibody oran antigen binding fragment thereof that binds to CD40, wherein theantibody alone is capable of causing B cell proliferation of at least10% of the B cells. In one aspect, the percentage of B cells thatproliferate is at least 15%, 20%, 25%, 28%, 30% or 35%. In one aspect,the antibody comprises at least one variable domain having 90% sequenceidentity with at least one antibody light chain variable region of SEQID NOS: 2, 4, 5 or 7; and at least one variable domain having 90%sequence identity with one antibody heavy chain variable region of SEQID NOS: 1, 3 or 7. In another aspect, the antibody comprises thepolypeptide sequence of SEQ ID NOS: 1, 3 or 6, the polypeptide sequenceof SEQ ID NOS: 2, 4, 5, or 7, or both. In another aspect, the antibodyis produced by a hybridoma selected from anti-CD40_12E12.3F3 (ATCCAccession No. PTA-9854), anti-CD40_12B4.2C10 (ATCC Submission No. HS446,Accession No. PTA-10653), and anti-CD40_11B6.1C3 (ATCC Submission No.HS440, Accession No. PTA-10652). In another aspect, the antibody ishumanized. In another aspect, antibody alone is capable of causingdendritic cells to secrete at least one of IL-6, MIP-1a, IL-12p40 orTNFalpha without prior activation of the dendritic cells. In anotheraspect, the antibody is capable of causing dendritic cells activatedwith GM-CSF and Interferon alpha to secrete at least one of IL-6,MIP-1a, IP-10, IL-10 or IL-12p40.

DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 shows protein A affinity recombinant antibodies fused to variousHIV peptides (lanes 1 to 5) secreted from transfected 293F cells,analyzed by reducing SDS-PAGE and Coomassie Brilliant Blue staining.

FIG. 2 shows protein A affinity purified recombinant antibodies fused tovarious HIV peptides (Lanes 1 and 2) secreted from transfected 293Fcells, then analyzed by reducing SDS-PAGE and Coomassie Brilliant Bluestaining.

FIG. 3 shows protein A affinity purified recombinant antibodies fused tovarious HIV peptide strings (Lanes 1 to 5) secreted from transfected293F cells, then analyzed by reducing SDS.PAGE and Coomassie BrilliantBlue staining.

FIG. 4 shows protein A affinity purified recombinant antibodies fused tovarious HIV peptide strings (Lanes 1 to 6) secreted from transfected293F cells, then analyzed by reducing SDS.PAGE and Coomassie BrilliantBlue staining.

FIG. 5 describes the protocol used in vitro to assay the potency ofαCD40.LIPO5 HIV peptide fusion recombinant antibody (αCD40.LIPO5 rAb) toelicit the expansion of antigen-specific T cells in the context of aPBMC culture.

FIG. 6A-C shows HIV peptide-specific IFN-γ production in PBMCs from HIVpatients incubated with various concentrations of anti-CD40.LIPO5peptide string vaccine. C is the control group, which received novaccine, and defines the baseline response of the culture to eachpeptide.

FIG. 7 is a summary of αCD40.LIPO5 peptide vaccine responses against the5 peptide regions from 8 HIV patients.

FIG. 8A-B shows that the αCD40.LIPO5 HIV peptide vaccine elicitsexpansion of HIV peptide-specific T cells capable of secreting multiplecytokines—a desirable feature in a vaccine. FIG. 8A-B also shows thatthe αCD40.LIPO5 HIV peptide vaccine elicits gag253, nef66, nef116 andpol325 peptide-specific responses characterized by production ofmultiple cytokines (patient A5).

FIG. 9 shows the protocol for testing αCD40.LIPO5 HIV peptide vaccinefor its ability to direct the expansion of antigen-specific T cellsresulting from targeted uptake by DCs and presentation of peptideepitopes on their surface MHC complex.

FIG. 10A-B shows the cytokine secretion in response to HIV peptides fromDC-T cell co-cultures treated with various doses of αCD40.LIPO5 HIVpeptide vaccine (patient A10).

FIG. 11A-B shows PBMCs from patient A4 treated with the αCD40.LIPO5 HIVpeptide vaccine elicit expansion of antigen-specific T cells withspecificity to the gag253 region, but not to the flexible linkersequences.

FIG. 12A is the αCD40.LIPO5 HIV peptide vaccine heavy chain sequenceshowing flexible linker regions in bold, joining sequences underlinedand HIV peptide regions shaded in grey. FIG. 12A shows PBMCs frompatient A3 treated with the αCD40.LIPO5 HIV peptide vaccine elicitexpansion of antigen-specific T cells with specificities to the gag253,nef66, and nef116 regions, but not to the flexible linker sequences.FIGS. 12B-1 and 12B-2 shows HIV antigen-specific T cell responses evokedfrom HIV patient A17 PBMCs incubated with 30 nM of three different HIVSpeptide DC targeting vaccines. FIGS. 12C-1 and 12C-2 is a similar studyto that shown in FIGS. 12B-1 and 12B-2, except that the PBMCs are from adifferent HIV patient (A2). FIG. 12D shows 15 different HIV peptideresponses [5 peptide regions sampled in 3 patients], it was found thatthe anti-CD40.HIV5pep vaccine was superior to anti-DCIR.HIV5pep,anti-LOX-1.HIV5pep and non-LIPO5 mix for eliciting a broad range of HIVpeptide-specific CD8+ and CD4+ T responses.

FIG. 13 shows the internalization of anti-CD40 mAb:IL-4DC. IL-4DCs weretreated with 500 ng/ml of anti-CD40-Alexa 568.

FIG. 14 shows CD4 and CD8 T cell proliferation by DCs targeted withanti-CD40-HA1. 5×10e3 IFNDCs loaded with 2 ug/ml of anti-CD40-HA orcontrol Ig-HA1 were co-cultured with CFSE-labeled autologous CD4+ orCD8+ T cells (2×10e5) for 7 days. Cells were then stained with anti-CD4or anti-CD8 antibodies. Cell proliferation was tested by measuringCFSE-dilution.

FIG. 15 shows a titration of HA1 fusion protein on CD4+ T proliferation.IFNDCs (5K) loaded with fusion proteins were co-cultured withCFSE-labeled CD4+ T cells (200K) for 7 days.

FIG. 16 shows IFNDCs targeted with anti-CD40-HA1 activate HA1-specificCD4+ T cells. CD4+ T cells were re-stimulated with DCs loaded with 5 uMof indicated peptides, and then intracellular IFNγ was stained.

FIG. 17 shows IFNDCs targeted with anti-CD40-HA1 activate HA1-specificCD4+ T cells. CD4+ T cells were re-stimulated with DCs loaded withindicated peptides for 36 h, and then culture supernatant was analyzedfor measuring IFNγ.

FIG. 18 shows that targeting CD40 results in enhanced cross-priming ofMART-1 specific CD8+ T cells. IFNDCs (5K/well) loaded with fusionproteins were co-cultured with purified CD8+ T cells for 10 days. Cellswere stained with anti-CD8 and tetramer. Cells are from healthy donors(HLA-A*0201+).

FIG. 19 shows targeting CD40 results in enhanced cross-priming of MART-1specific CD8+ T cells (Summary of 8-repeated experiments using cellsfrom different healthy donors).

FIG. 20 shows CD8+ CTL induced with IFNDCs targeted withanti-CD40-MART-1 are functional. CD8+ T cells co-cultured with IFNDCstargeted with fusion proteins were mixed with T2 cells loaded with 10 uMpeptide epitope.

FIG. 21 shows CD8+ CTL induced with IFNDCs targeted with anti-CD40-FluM1 are functional. CD8+ T cells co-cultured with IFNDCs targeted withfusion proteins were mixed with T2 cells loaded with 1.0 nM peptideepitope.

FIG. 22 shows an outline of protocol to test the ability a vaccinecomposed of anti-CD4012E12 linked to PSA (prostate specific antigen) toelicit the expansion from a naïve T cell population. PSA-specific CD4+ Tcells corresponding to a broad array of PSA epitopes. Briefly, DCsderived by culture with IFNα and GM-CSF of monocytes from a healthydonor are incubated with the vaccine. The next day, cells are placed infresh medium and pure CD4+ T cells from the same donor are added.Several days later, PSA peptides are added and, after four hours,secreted gamma-IFN levels in the culture supernatants are determined.

FIG. 23 shows that many PSA peptides elicit potent gamma-IFN-productionresponses indicating that anti-CD4012E12 and similar anti-CD40 agentscan efficiently deliver antigen to DCs, resulting in the priming ofimmune responses against multiple epitopes of the antigen.

FIG. 24 shows DCs targeted with anti-CD40-PSA induce PSA-specific CD8+ Tcell responses. IFNDCs were targeted with 1 ug mAb fusion protein withPSA. Purified autologous CD8+ T cells were co-cultured for 10 days.Cells were stained with anti-CD8 and PSA (KLQCVDLH—SEQ ID NO:131)-tetramer. Cells are from a HLA-A*0201 positive healthy donor. Theresults demonstrate that anti-CD40 effectively deliver PSA to the DCs,which in turn elicit the expansion of PSA-specific CD8+ T cells.

FIG. 25 a scheme (left) and the IFN-γ production by T cells of the poolsof peptides and control for Donor 2. 5×10e3 IFNDCs loaded with 2 ug/mlof anti-CD40-Cyclin D1 were co-cultured with purified autologous CD4+ Tcells (2×10e5) for 8 days. Cells were then re-stimulated with with 5 uMof individual peptides derived from CyclinD1 for 5 h in the presence ofBrefeldin A. Cells were stained for measuring intracellular IFNγexpression.

FIG. 26 shows a peptide scan and IFN-γ production by T cells obtainedfrom the pools of peptides shown in FIG. 25 and control for Donor 2.5×10e3 IFNDCs loaded with 2 ug/ml of anti-CD40-Cyclin D1 wereco-cultured with purified autologous CD4+ T cells (2×10e5) for 8 days.Cells were then re-stimulated with 5 uM of individual peptides derivedfrom CyclinD1 for 5 h in the presence of Brefeldin A. Cells were stainedfor measuring intracellular IFN-γ expression.

FIG. 27 shows the expression and construct design for anti-CD40-MART-1peptide antibodies.

FIG. 28 is a summary of the CD4⁺ and CD8⁺ immunodominant epitopes forMART-1.

FIG. 29 shows the expression and construct design for anti-CD40-gp100peptide antibodies.

FIG. 30 shows the design for additional anti-CD40-gp100 peptideantibodies.

FIG. 31 shows the expression and construct design for additionalanti-CD40-gp100 peptide antibodies.

FIG. 32 is a summary of the CD4⁺ and CD8⁺ immunodominant epitopes forgp100.

FIG. 33 shows the expression and construct design for additionalanti-CD40-gp100 peptide antibodies.

FIG. 34 shows the results obtained with the various antibodies using anassay that detects signaling via CD40 ligation—read out as cell death.

FIG. 35 shows the binding of various constructs when the antibody hasbeen made into a fusion protein with doc and then captures.

FIGS. 36 and 37 compare cytokine production with our without theaddition of GM-CSF and IFNα (FIG. 36 A-D), and soluble antibodies alone(FIG. 37A-D) incubated with the DCs for 24 hours.

FIG. 38A-B demonstrates the effect of various concentrations ofanti-CD40 antibodies of the present invention on B cell proliferation.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The invention includes also variants and other modification of anantibody (or “Ab”) of fragments thereof, e.g., anti-CD40 fusion protein(antibody is used interchangeably with the term “immunoglobulin”). Asused herein, the term “antibodies or fragments thereof,” includes wholeantibodies or fragments of an antibody, e.g., Fv, Fab, Fab′, F(ab′)₂,Fc, and single chain Fv fragments (ScFv) or any biologically effectivefragments of an immunoglobulins that binds specifically to, e.g., CD40.Antibodies from human origin or humanized antibodies have lowered or noimmunogenicity in humans and have a lower number or no immunogenicepitopes compared to non-human antibodies. Antibodies and theirfragments will generally be selected to have a reduced level or noantigenicity in humans.

As used herein, the terms “Ag” or “antigen” refer to a substance capableof either binding to an antigen binding region of an immunoglobulinmolecule or of eliciting an immune response, e.g., a T cell-mediatedimmune response by the presentation of the antigen on MajorHistocompatibility Antigen (MHC) cellular proteins. As used herein,“antigen” includes, but is not limited to, antigenic determinants,haptens, and immunogens which may be peptides, small molecules,carbohydrates, lipids, nucleic acids or combinations thereof. Theskilled immunologist will recognize that when discussing antigens thatare processed for presentation to T cells, the term “antigen” refers tothose portions of the antigen (e.g., a peptide fragment) that is a Tcell epitope presented by MHC to the T cell receptor. When used in thecontext of a B cell mediated immune response in the form of an antibodythat is specific for an “antigen”, the portion of the antigen that bindsto the complementarity determining regions of the variable domains ofthe antibody (light and heavy) the bound portion may be a linear orthree-dimensional epitope. In the context of the present invention, theterm antigen is used on both contexts, that is, the antibody is specificfor a protein antigen (CD40), but also carries one or more peptideepitopes for presentation by MHC to T cells. In certain cases, theantigens delivered by the vaccine or fusion protein of the presentinvention are internalized and processed by antigen presenting cellsprior to presentation, e.g., by cleavage of one or more portions of theantibody or fusion protein.

As used herein, the term “antigenic peptide” refers to that portion of apolypeptide antigen that is specifically recognized by either B-cells orT-cells. B-cells respond to foreign antigenic determinants via antibodyproduction, whereas T-lymphocytes are the mediate cellular immunity.Thus, antigenic peptides are those parts of an antigen that arerecognized by antibodies, or in the context of an MHC, by T-cellreceptors.

As used herein, the term “epitope” refers to any protein determinantcapable of specific binding to an immunoglobulin or of being presentedby a Major Histocompatibility Complex (MHC) protein (e.g., Class I orClass II) to a T-cell receptor. Epitopic determinants are generallyshort peptides 5-30 amino acids long that fit within the groove of theMHC molecule that presents certain amino acid side groups toward the Tcell receptor and has certain other residues in the groove, e.g., due tospecific charge characteristics of the groove, the peptide side groupsand the T cell receptor. Generally, an antibody specifically binds to anantigen when the dissociation constant is 1 mM, 100 nM or even 10 nM.

As used herein, the term “vector” is used in two different contexts.When using the term “vector” with reference to a vaccine, a vector isused to describe a non-antigenic portion that is used to direct ordeliver the antigenic portion of the vaccine. For example, an antibodyor fragments thereof may be bound to or form a fusion protein with theantigen that elicits the immune response. For cellular vaccines, thevector for delivery and/or presentation of the antigen is the antigenpresenting cell, which is delivered by the cell that is loaded withantigen. In certain cases, the cellular vector itself may also processand present the antigen(s) to T cells and activate an antigen-specificimmune response. When used in the context of nucleic acids, a “vector”refers a construct, which is capable of delivering, and preferablyexpressing, one or more genes or polynucleotide sequences of interest ina host cell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, DNA or RNA expressionvectors associated with cationic condensing agents, DNA or RNAexpression vectors encapsulated in liposomes, and certain eukaryoticcells, such as producer cells.

As used herein, the terms “stable” and “unstable” when referring toproteins is used to describe a peptide or protein that maintains itsthree-dimensional structure and/or activity (stable) or that losesimmediately or over time its three-dimensional structure and/or activity(unstable). As used herein, the term “insoluble” refers to thoseproteins that when produced in a cell (e.g., a recombinant proteinexpressed in a eukaryotic or prokaryotic cell or in vitro) are notsoluble in solution absent the use of denaturing conditions or agents(e.g., heat or chemical denaturants, respectively). The antibody orfragment thereof and the linkers taught herein have been found toconvert antibody fusion proteins with the peptides from insoluble and/orunstable into proteins that are stable and/or soluble. Another exampleof stability versus instability is when the domain of the protein with astable conformation has a higher melting temperature (T_(m)) than theunstable domain of the protein when measured in the same solution. Adomain is stable compared to another domain when the difference in theT_(m) is at least about 2° C., more preferably about 4° C., still morepreferably about 7° C., yet more preferably about 10° C., even morepreferably about 15° C., still more preferably about 20° C., even stillmore preferably about 25° C., and most preferably about 30° C., whenmeasured in the same solution.

As used herein, “polynucleotide” or “nucleic acid” refers to a strand ofdeoxyribonucleotides or ribonucleotides in either a single- or adouble-stranded form (including known analogs of natural nucleotides). Adouble-stranded nucleic acid sequence will include the complementarysequence. The polynucleotide sequence may encode variable and/orconstant region domains of immunoglobulin that are formed into a fusionprotein with one or more linkers. For use with the present invention,multiple cloning sites (MCS) may be engineered into the locations at thecarboxy-terminal end of the heavy and/or light chains of the antibodiesto allow for in-frame insertion of peptide for expression between thelinkers. As used herein, the term “isolated polynucleotide” refers to apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof. By virtue of its origin the “isolated polynucleotide” (1) isnot associated with all or a portion of a polynucleotide in which the“isolated polynucleotides” are found in nature, (2) is operably linkedto a polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. The skilled artisan willrecognize that to design and implement a vector can be manipulated atthe nucleic acid level by using techniques known in the art, such asthose taught in Current Protocols in Molecular Biology, 2007 by JohnWiley and Sons, relevant portions incorporated herein by reference.Briefly, the encoding nucleic acid sequences can be inserted usingpolymerase chain reaction, enzymatic insertion of oligonucleotides orpolymerase chain reaction fragments in a vector, which may be anexpression vector. To facilitate the insertion of inserts at the carboxyterminus of the antibody light chain, the heavy chain, or both, amultiple cloning site (MCS) may be engineered in sequence with theantibody sequences.

As used herein, the term “polypeptide” refers to a polymer of aminoacids and does not refer to a specific length of the product; thus,peptides, oligopeptides, and proteins are included within the definitionof polypeptide. This term also does not refer to or exclude postexpression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, polypeptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring. The term “domain,” or “polypeptide domain”refers to that sequence of a polypeptide that folds into a singleglobular region in its native conformation, and that may exhibitdiscrete binding or functional properties.

A polypeptide or amino acid sequence “derived from” a designated nucleicacid sequence refers to a polypeptide having an amino acid sequenceidentical to that of a polypeptide encoded in the sequence, or a portionthereof wherein the portion consists of at least 3-5 amino acids,preferably at least 4-7 amino acids, more preferably at least 8-10 aminoacids, and even more preferably at least 11-15 amino acids, or which isimmunologically identifiable with a polypeptide encoded in the sequence.This terminology also includes a polypeptide expressed from a designatednucleic acid sequence.

As used herein, “pharmaceutically acceptable carrier” refers to anymaterial that when combined with an immunoglobulin (Ig) fusion proteinof the present invention allows the Ig to retain biological activity andis generally non-reactive with the subject's immune system. Examplesinclude, but are not limited to, standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such as anoil/water emulsion, and various types of wetting agents. Certaindiluents may be used with the present invention, e.g., for aerosol orparenteral administration, that may be phosphate buffered saline ornormal (0.85%) saline.

An antibody for use with the present invention comprises at least thevariable region of anti-CD40_12E12.3F3 (ATCC Accession No. PTA-9854),anti-CD40_12B4.2C10 (Deposit No. HS446, ATCC Accession No. PTA-10653),and anti-CD40_11B6.1C3 (Deposit No. HS440, ATCC Accession No.PTA-10652). The hybridoma cell lines have been deposited with theAmerican Type Culture Collection, the CD40_12E12.3F3 producing hybridoma(ATCC Accession No. PTA-9854) having been deposited on Feb. 26, 2009 andthe anti-CD40_12B4.2C10 (Deposit No. HS446, ATCC Accession No.PTA-10653), and anti-CD40_11B6.1C3 (Deposit No. HS440, ATCC AccessionNo. PTA-10652) producing hybridomas having been deposited on Feb. 17,2010. Contact information for the American Type Culture Collection isthe following: IP, Licensing and Services; 10801 University Boulevard;Manassas, Va. 20110-2209 USA.

The invention provides an CD40 binding molecule comprising at least oneimmunoglobulin light chain variable domain (VL) which comprises insequence hypervariable regions CDR1L, CDR2L and CDR3L, the CDR1L havingthe amino acid sequence SASQGISNYLN (SEQ ID NO.:41) the CDR2L having theamino acid sequence YTSILHS (SEQ ID NO.:42) and the CDR3L having theamino acid sequence QQFNKLPPT (SEQ ID NO.:43) and direct equivalentsthereof for the anti-CD40_11B6.1C3, or the anti-CD40_12B4.2C10antibodies.

Accordingly the invention provides an CD40 binding molecule whichcomprises an antigen binding site comprising at least one immunoglobulinheavy chain variable domain (VH) which comprises in sequencehypervariable regions CDR1H, CDR2H and CDR3H, the CDR1H having the aminoacid sequence GFTFSDYYMY (SEQ ID NO.:44), the CDR2H having the aminoacid sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:45), and the CDR3H havingthe amino acid sequence RGLPFHAMDY (SEQ ID NO.:46), and directequivalents thereof the anti-CD40_11B6.1C3, or the anti-CD40_12B4.2C10antibodies.

In one aspect the invention provides a single domain CD40 bindingmolecule comprising an isolated immunoglobulin light chain comprising aheavy chain variable domain (VL) as defined above. In another aspect theinvention provides a single domain CD40 binding molecule comprising anisolated immunoglobulin heavy chain comprising a heavy chain variabledomain (VH) as defined above.

In another aspect the invention also provides an CD40 binding moleculecomprising both heavy (VH) and light chain (VL) variable domains inwhich the CD40 binding molecule comprises at least one antigen bindingsite comprising: a) an immunoglobulin heavy chain variable domain (VL)which comprises in sequence hypervariable regions CDR1L, CDR2L andCDR3L, the CDR1L having the amino acid sequence SASQGISNYLN (SEQ IDNO.:41), the CDR2L having the amino acid sequence YTSILHS (SEQ IDNO.:42), and the CDR3L having the amino acid sequence QQFNKLPPT (SEQ IDNO.:43), and b) an immunoglobulin light chain variable domain (VH) whichcomprises in sequence hypervariable regions CDR1H, CDR2H and CDR3H, theCDR1H having the amino acid sequence GFTFSDYYMY (SEQ ID NO.:44), theCDR2′ having the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:45),and the CDR3H having the amino acid sequence RGLPFHAMDY (SEQ ID NO.:46)and direct equivalents thereof the anti-CD40_11B6.1C3, or theanti-CD40_12B4.2C10 antibodies.

Unless otherwise indicated, any polypeptide chain is herein described ashaving an amino acid sequence starting at the N-terminal end and endingat the C-terminal end. When the antigen binding site comprises both theVH and VL domains, these may be located on the same polypeptide moleculeor, preferably, each domain may be on a different chain, the VH domainbeing part of an immunoglobulin heavy chain or fragment thereof and theVL being part of an immunoglobulin light chain or fragment thereof.

As used herein, the term “CD40 binding molecule” refers to any moleculecapable of binding to the CD40 antigen either alone or associated withother molecules having one or more the V_(L) and V_(H) CDRs taughtherein, in some cases 2, 3, 4, 5, or all 6 CDRs. The binding reactionmay be shown by standard methods (qualitative assays) including, forexample, a bioassay for determining by blocking the binding of othermolecules to CD40 or any kind of binding or activity assays (e.g.,activation, reduction or modulation of an immune response), withreference to a negative control test in which an antibody of unrelatedspecificity but of the same isotype, e.g., an anti-CD25 or anti-CD80antibody, is used.

The present invention may also be made into a single chain antibodyhaving the variable domains of the heavy and light chains of an antibodycovalently bound by a peptide linker usually including from 10 to 30amino acids, preferably from 15 to 25 amino acids. Therefore, such astructure does not include the constant part of the heavy and lightchains and it is believed that the small peptide spacer should be lessantigenic than a whole constant part.

As used herein, the term “chimeric antibody” refers to an antibody inwhich the constant regions of heavy or light chains or both are of humanorigin while the variable domains of both heavy and light chains are ofnon-human (e.g., mouse, hamster or rat) origin or of human origin butderived from a different human antibody.

As used herein, the term “CDR-grafted antibody” refers to an antibody inwhich the hypervariable complementarity determining regions (CDRs) arederived from a donor antibody, such as a non-human (e.g., mouse)antibody or a different human antibody, while all or substantially allthe other parts of the immunoglobulin (e.g., the conserved regions ofthe variable domains, i.e., framework regions), are derived from anacceptor antibody (in the case of a humanized antibody—an antibody ofhuman origin). A CDR-grafted antibody may include a few amino acids ofthe donor sequence in the framework regions, for instance in the partsof the framework regions adjacent to the hypervariable regions.

As used herein, the term “human antibody” refers to an antibody in whichthe constant and variable regions of both the heavy and light chains areall of human origin, or substantially identical to sequences of humanorigin, not necessarily from the same antibody and includes antibodiesproduced by mice in which the mouse, hamster or rat immunoglobulinvariable and constant part genes have been replaced by their humancounterparts, e.g. as described in general terms in EP 0546073 B1, U.S.Pat. No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126,U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,661,016, U.S. Pat. No.5,770,429, EP 0 438474 B1 and EP 0 463151 B1, relevant portionsincorporated herein by reference.

The CD40 binding molecule of the invention can be a humanized antibodythat comprises the CDRs obtained from the anti-CD40_12E12.3F3, theanti-CD40_11B6.1C3, or the anti-CD40_12B4.2C10 antibodies. One exampleof a chimeric antibody includes the variable domains of both heavy andlight chains are of human origin, for instance those variable domains ofthe anti-CD40_12E12.3F3 antibody that are part of SEQ ID NO.: 1 and SEQID NO.: 2, anti-CD40_12B4.2C10 in SEQ ID NO.: 3 and SEQ ID NO.: 4 or SEQID NO.: 5; and/or anti-CD40_11B6.1C3, SEQ ID NO.: 6 and SEQ ID NO.: 7,or combination thereof. The constant region domains preferably alsocomprise suitable human constant region domains, for instance asdescribed in “Sequences of Proteins of Immunological Interest”, Kabat E.A. et al, US Department of Health and Human Services, Public HealthService, National Institute of Health. The nucleic acid sequences can befound in, e.g., SEQ ID NOS.: 8 and 9.

Hypervariable regions may be associated with any kind of frameworkregions, e.g., of human origin. Suitable framework regions weredescribed Kabat E. A. One heavy chain framework is a heavy chainframework, for instance that of anti-CD40_12E12.3F3 antibody that arepart of SEQ ID NO.: 2; anti-CD40_12B4.2C10—SEQ ID NO.: 4 or SEQ ID NO.:5, and/or anti-CD40_11B6.1C3—SEQ ID NO.: 7, or combination thereof,e.g., FR1_(L), FR2_(L), FR3_(L) and FR4_(L) regions. In a similarmanner, SEQ ID NO. 1 shows the anti-CD40_12E12.3F3 (or the equivalentsfor anti-CD40_12B4.2C10 and anti-CD40_11B6.1C3, SEQ ID NOS.: 3 and 6,respectively) heavy chain framework that includes the sequence ofFR1_(H), FR2_(H), FR3_(H) and FR4_(H) regions. The CDRs may be added toa human antibody framework, such as those described in U.S. Pat. No.7,456,260, issued to Rybak, et al., which teach new human variable chainframework regions and humanized antibodies comprising the frameworkregions, relevant portions and framework sequences incorporated hereinby reference. To accomplish the engraftment at a genetic level, thepresent invention also includes the underlying nucleic acid sequencesfor the V_(L) AND V_(H) regions as well as the complete antibodies andthe humanized versions thereof. The nucleic acid sequences of thepresent invention include SEQ ID NOS.: 8 and 9, which are the anti-CD40antibody light and the heavy chains, respectively, as well as thosenucleic acid sequences that include variable codon usage for the sameamino acid sequences and conservative variations thereof having 85, 90,95 or 100% sequence identity at the nucleic or amino acid level.Likewise, the CDRs may have 85, 90, 95 or 100% sequence identity at thenucleic or amino acid level, individually, in groups or 2, 3, 4 or 5 orall together.

Monoclonal antibodies raised against a protein naturally found in allhumans are typically developed in a non-human system e.g. in mice, andas such are typically non-human proteins. As a direct consequence ofthis, a xenogenic antibody as produced by a hybridoma, when administeredto humans, elicits an undesirable immune response that is predominantlymediated by the constant part of the xenogenic immunoglobulin.Xenogeneic antibodies tend to elicit a host immune response, therebylimiting the use of such antibodies as they cannot be administered overa prolonged period of time. Therefore, it is particularly useful to usesingle chain, single domain, chimeric, CDR-grafted, or especially humanantibodies that are not likely to elicit a substantial allogenicresponse when administered to humans. The present invention includesantibodies with minor changes in an amino acid sequence such asdeletion, addition or substitution of one, a few or even several aminoacids which are merely allelic forms of the original protein havingsubstantially identical properties.

The inhibition of the binding of CD40 to its receptor may beconveniently tested in various assays including such assays aredescribed hereinafter in the text. By the term “to the same extent” ismeant that the reference and the equivalent molecules exhibit, on astatistical basis, essentially identical CD40 binding inhibition curvesin one of the assays referred to above. For example, the assay used maybe an assay of competitive inhibition of binding of CD40 by the bindingmolecules of the invention.

Generally, the human anti-CD40 antibody comprises at least: (a) onelight chain which comprises a variable domain having an amino acidsequence substantially identical to that shown in SEQ ID NO.: 1 startingwith the amino acid at position 1 and ending with the amino acid atposition 107 and the constant part of a human light chain; and (b) oneheavy chain which comprises a variable domain having an amino acidsequence substantially identical to that shown in SEQ ID NO. 2 and theconstant part of a human heavy chain. The constant part of a human heavychain may be of the γ1, γ2, γ3, γ4, μ, β2, or δ or ε type, preferably ofthe γ-type, whereas the constant part of a human light chain may be ofthe κ or λ type (which includes the λ₁, λ₂ and λ₃ subtypes) but ispreferably of the κ type. The amino acid sequences of the generallocations of the variable and constant domains are well known in the artand generally follow the Kabat nomenclature.

A CD40 binding molecule of the invention may be produced by recombinantDNA techniques. In view of this, one or more DNA molecules encoding thebinding molecule must be constructed, placed under appropriate controlsequences and transferred into a suitable host organism for expression.

In a very general manner, there are accordingly provided: (i) DNAmolecules encoding a single domain CD40 binding molecule of theinvention, a single chain CD40 binding molecule of the invention, aheavy or light chain or fragments thereof of a CD40 binding molecule ofthe invention; and (ii) the use of the DNA molecules of the inventionfor the production of a CD40 binding molecule of the invention byrecombinant methods.

The present state of the art is such that the skilled worker in the artcan synthesize the DNA molecules of the invention given the informationprovided herein, i.e., the amino acid sequences of the hypervariableregions and the DNA sequences coding for them. A method for constructinga variable domain gene is for example described in EPA 239 400, relevantportions incorporated herein by reference. Briefly, a gene encoding avariable domain of a MAb is cloned. The DNA segments encoding theframework and hypervariable regions are determined and the DNA segmentsencoding the hypervariable regions are removed so that the DNA segmentsencoding the framework regions are fused together with suitablerestriction sites at the junctions. The restriction sites may begenerated at the appropriate positions by mutagenesis of the DNAmolecule by standard procedures. Double stranded synthetic CDR cassettesare prepared by DNA synthesis according to the sequences given in SEQ IDNO.: 1 and 3 or 2 and 4 (amino acid and nucleic acid sequences,respectively). These cassettes are often provided with sticky ends sothat they can be ligated at the junctions of the framework.

It is not necessary to have access to the mRNA from a producinghybridoma cell line in order to obtain a DNA construct coding for theCD40 binding molecules of the invention. For example, PCT application WO90/07861 gives full instructions for the production of an antibody byrecombinant DNA techniques given only written information as to thenucleotide sequence of the gene, relevant portions incorporated hereinby reference. Briefly, the method comprises the synthesis of a number ofoligonucleotides, their amplification by the PCR method, and theirsplicing to give the desired DNA sequence.

Expression vectors comprising a suitable promoter or genes encodingheavy and light chain constant parts are publicly available. Thus, oncea DNA molecule of the invention is prepared it may be convenientlytransferred in an appropriate expression vector. DNA molecules encodingsingle chain antibodies may also be prepared by standard methods, forexample, as described in WO 88/1649. In view of the foregoing, nohybridoma or cell line deposit is necessary to comply with the criteriaof sufficiency of description.

For example, first and second DNA constructs are made that bindspecifically to CD40. Briefly, a first DNA construct encodes a lightchain or fragment thereof and comprises a) a first part which encodes avariable domain comprising alternatively framework and hypervariableregions, the hypervariable regions being in sequence CDR1_(L), CDR2_(L)and CDR3_(L) the amino acid sequences of which are shown in SEQ ID NO.:1; this first part starting with a codon encoding the first amino acidof the variable domain and ending with a codon encoding the last aminoacid of the variable domain, and b) a second part encoding a light chainconstant part or fragment thereof which starts with a codon encoding thefirst amino acid of the constant part of the heavy chain and ends with acodon encoding the last amino acid of the constant part or fragmentthereof, followed by a stop codon.

The first part encodes a variable domain having an amino acid sequencesubstantially identical to the amino acid sequence as shown in SEQ IDNO.: 1, 2, 3, 4, 5, 6 or 7. A second part encodes the constant part of ahuman heavy chain, more preferably the constant part of the human γ1chain. This second part may be a DNA fragment of genomic origin(comprising introns) or a cDNA fragment (without introns).

The second DNA construct encodes a heavy chain or fragment thereof andcomprises a) a first part which encodes a variable domain comprisingalternatively framework and hypervariable regions; the hypervariableregions being CDR1_(H) and optionally CDR2_(H) and CDR3_(H), the aminoacid sequences of which are shown in SEQ ID NO. 2; this first partstarting with a codon encoding the first amino acid of the variabledomain and ending with a codon encoding the last amino acid of thevariable domain, and b) a second part encoding a heavy chain constantpart or fragment thereof which starts with a codon encoding the firstamino acid of the constant part of the light chain and ends with a codonencoding the last amino acid of the constant part or fragment thereoffollowed by a stop codon.

The first part encodes a variable domain having an amino acid sequencesubstantially identical to the amino acid sequence as shown in SEQ IDNO. 2. The first part has the nucleotide sequence as shown in SEQ ID NO.2 starting with the nucleotide at position 1 and ending with thenucleotide at position 321. Also preferably the second part encodes theconstant part of a human light chain, more preferably the constant partof the human κ chain.

The invention also includes CD40 binding molecules in which one or moreof the residues of CDR1_(L), CDR2_(L), CDR3_(L), CDR1_(H), CDR2_(H) orCDR3_(H) or the frameworks, typically only a few (e.g. FR1-4_(L) or H),are changed from the residues shown in SEQ ID NO. 37 and SEQ ID NO. 38;by, e.g., site directed mutagenesis of the corresponding DNA sequences.The invention includes the DNA sequences coding for such changed CD40binding molecules. In particular the invention includes a CD40 bindingmolecules in which one or more residues of CDR1_(L), CDR2_(L) and/orCDR3_(L) have been changed from the residues shown in SEQ ID NO. 37 andone or more residues of CDR1_(H), CDR2_(H) and/or CDR3_(H) have beenchanged from the residues shown in SEQ ID NO. 38, or the equivalentsfrom SEQ ID NOS.: 1, 3 and 6.

Each of the DNA constructs are placed under the control of suitablecontrol sequences, in particular under the control of a suitablepromoter. Any kind of promoter may be used, provided that it is adaptedto the host organism in which the DNA constructs will be transferred forexpression. However, if expression is to take place in a mammalian cell,an immunoglobulin gene promoter may be used in B cells. The first andsecond parts may be separated by an intron, and, an enhancer may beconveniently located in the intron between the first and second parts.The presence of such an enhancer that is transcribed but not translated,may assist in efficient transcription. In particular embodiments thefirst and second DNA constructs comprise the enhancer of, e.g., a heavychain human gene.

The desired antibody may be produced in a cell culture or in atransgenic animal. A suitable transgenic animal may be obtainedaccording to standard methods that include micro injecting into eggs thefirst and second DNA constructs placed under suitable control sequencestransferring the so prepared eggs into appropriate pseudo-pregnantfemales and selecting a descendant expressing the desired antibody.

The invention also provides an expression vector able to replicate in aprokaryotic or eukaryotic cell line, which comprises at least one of theDNA constructs above described. Each expression vector containing a DNAconstruct is then transferred into a suitable host organism. When theDNA constructs are separately inserted on two expression vectors, theymay be transferred separately, i.e. one type of vector per cell, orco-transferred, this latter possibility being preferred. A suitable hostorganism may be a bacterium, a yeast or a mammalian cell line, thislatter being preferred. More preferably, the mammalian cell line is oflymphoid origin, e.g., a myeloma, hybridoma or a normal immortalizedB-cell, which conveniently does not express any endogenous antibodyheavy or light chain.

When the antibody chains are produced in a cell culture, the DNAconstructs must first be inserted into either a single expression vectoror into two separate but compatible expression vectors, the latterpossibility being preferred. For expression in mammalian cells it ispreferred that the coding sequence of the CD40 binding molecule isintegrated into the host cell DNA within a locus which permits or favorshigh level expression of the CD40 binding molecule.

In a further aspect of the invention there is provided a process for theproduct of a CD40 binding molecule that comprises: (i) culturing anorganism which is transformed with an expression vector as definedabove; and (ii) recovering the CD40 binding molecule from the culture.

In accordance with the present invention it has been found that theanti-CD40_12E12.3F3, anti-CD40_12B4.2C10 and/or anti-CD40_11B6.1C3antibody appears to have binding specificity for human CD40. It istherefore most surprising that antibodies to this epitope, e.g. theanti-CD40_12E12.3F3, anti-CD40_12B4.2C10 and/or anti-CD40_11B6.1C3antibody, are capable of delivering antigen efficiently into dendriticcells (DCs). Antibodies, in particular chimeric and CDR-graftedantibodies and especially human antibodies, which have bindingspecificity for the antigenic epitope of mature human CD40; and use ofsuch antibodies for DC antigen loading are novel and are included withinthe scope of the present invention.

To use the anti-CD40 antibody of the present invention for treatmentindications, the appropriate dosage will, of course, vary dependingupon, for example, the antibody disclosed herein to be employed, thehost, the mode of administration and the nature and severity of thecondition being treated. However, in prophylactic use, satisfactoryresults are generally found at dosages from about 0.05 mg to about 10 mgper kilogram body weight more usually from about 0.1 mg to about 5 mgper kilogram body weight. The frequency of dosing for prophylactic useswill normally be in the range from about once per week up to about onceevery 3 months, more usually in the range from about once every 2 weeksup to about once every 10 weeks, e.g., once every 4 to 8 weeks. Theanti-CD40 antibody of the present can be administered parenterally,intravenously, e.g., into the antecubital or other peripheral vein,intramuscularly, or subcutaneously.

Pharmaceutical compositions of the invention may be manufactured inconventional manner, e.g., in a lyophilized form. For immediateadministration it is dissolved in a suitable aqueous carrier, forexample sterile water for injection or sterile buffered physiologicalsaline. If it is considered desirable to make up a solution of largervolume for administration by infusion rather as a bolus injection, it isadvantageous to incorporate human serum albumin or the patient's ownheparinized blood into the saline at the time of formulation. Thepresence of an excess of such physiologically inert protein preventsloss of antibody by adsorption onto the walls of the container andtubing used with the infusion solution. If albumin is used, a suitableconcentration is from 0.5 to 4.5% by weight of the saline solution.

One embodiment of the present invention provides an immunoconjugatecomprising a humanized antibody of the invention, e.g., a humanizedanti-CD40 antibody, linked to one or more effector molecules, antigen(s)and/or a detectable label(s). Preferably, the effector molecule is atherapeutic molecule such as, for example, one or more peptides thatcomprise one or more T cell epitopes, a toxin, a small molecule, acytokine or a chemokine, an enzyme, or a radiolabel.

Exemplary toxins include, but are not limited to, Pseudomonas exotoxinor diphtheria toxin. Examples of small molecules include, but are notlimited to, chemotherapeutic compounds such as taxol, doxorubicin,etoposide, and bleiomycin. Exemplary cytokines include, but are notlimited to, IL-1, IL-2, IL-4, IL-5, IL-6, and IL-12, IL-17, and IL-25.Exemplary enzymes include, but are not limited to, RNAses, DNAses,proteases, kinases, and caspases. Exemplary radioisotopes include, butare not limited to, ³²P and ¹²⁵I.

As used herein, the term “epitope” refers to a molecule or substancecapable of stimulating an immune response. In one example, epitopesinclude but are not limited to a polypeptide and a nucleic acid encodinga polypeptide, wherein expression of the nucleic acid into a polypeptideis capable of stimulating an immune response when the polypeptide isprocessed and presented on a Major Histocompatibility Complex (MHC)molecule. Generally, epitopes include peptides presented on the surfaceof cells non-covalently bound to the binding groove of Class I or ClassII MHC, such that they can interact with T cell receptors and therespective T cell accessory molecules.

Proteolytic Processing of Antigens. Epitopes that are displayed by MHCon antigen presenting cells are cleavage peptides or products of largerpeptide or protein antigen precursors. For MHC I epitopes, proteinantigens are often digested by proteasomes resident in the cell.Intracellular proteasomal digestion produces peptide fragments of about3 to 23 amino acids in length that are then loaded onto the MHC protein.Additional proteolytic activities within the cell, or in theextracellular milieu, can trim and process these fragments further.Processing of MHC Class II epitopes generally occurs via intracellularproteases from the lysosomal/endosomal compartment. The presentinvention includes, in one embodiment, pre-processed peptides that areattached to the anti-CD40 antibody (or fragment thereof) that directsthe peptides against which an enhanced immune response is soughtdirectly to antigen presenting cells.

To identify epitopes potentially effective as immunogenic compounds,predictions of MHC binding alone are useful but often insufficient. Thepresent invention includes methods for specifically identifying theepitopes within antigens most likely to lead to the immune responsesought for the specific sources of antigen presenting cells andresponder T cells.

The present invention allows for a rapid and easy assay for theidentification of those epitopes that are most likely to produce thedesired immune response using the patient's own antigen presenting cellsand T cell repertoire. The compositions and methods of the presentinvention are applicable to any protein sequence, allowing the user toidentify the epitopes that are capable of binding to MHC and areproperly presented to T cells that will respond to the antigen.Accordingly, the invention is not limited to any particular target ormedical condition, but instead encompasses and MHC epitope(s) from anyuseful source.

As used herein, the term “veneered” refers to a humanized antibodyframework onto which antigen-binding sites or CDRs obtained fromnon-human antibodies (e.g., mouse, rat or hamster), are placed intohuman heavy and light chain conserved structural framework regions(FRs), for example, in a light chain or heavy chain polynucleotide to“graft” the specificity of the non-human antibody into a humanframework. The polynucleotide expression vector or vectors that expressthe veneered antibodies can be transfected mammalian cells for theexpression of recombinant human antibodies which exhibit the antigenspecificity of the non-human antibody and will undergo posttranslationalmodifications that will enhance their expression, stability, solubility,or combinations thereof.

Antigens.

Examples of viral antigens for use with the present invention include,but are not limited to, e.g., HIV, HCV, CMV, adenoviruses, retroviruses,picornaviruses, etc. Non-limiting example of retroviral antigens such asretroviral antigens from the human immunodeficiency virus (HIV) antigenssuch as gene products of the gag, pol, and env genes, the Nef protein,reverse transcriptase, and other HIV components; hepatitis viralantigens such as the S, M, and L proteins of hepatitis B virus, thepre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitisA, B, and C, viral components such as hepatitis C viral RNA; influenzaviral antigens such as hemagglutinin and neuraminidase and otherinfluenza viral components; measles viral antigens such as the measlesvirus fusion protein and other measles virus components; rubella viralantigens such as proteins E1 and E2 and other rubella virus components;rotaviral antigens such as VP7sc and other rotaviral components;cytomegaloviral antigens such as envelope glycoprotein B and othercytomegaloviral antigen components; respiratory syncytial viral antigenssuch as the RSV fusion protein, the M2 protein and other respiratorysyncytial viral antigen components; herpes simplex viral antigens suchas immediate early proteins, glycoprotein D, and other herpes simplexviral antigen components; varicella zoster viral antigens such as gpI,gpII, and other varicella zoster viral antigen components; Japaneseencephalitis viral antigens such as proteins E, M-E, M-E-NS1, NS1,NS1-NS2A, 80% E, and other Japanese encephalitis viral antigencomponents; rabies viral antigens such as rabies glycoprotein, rabiesnucleoprotein and other rabies viral antigen components. See FundamentalVirology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (RavenPress, New York, 1991) for additional examples of viral antigens. The atleast one viral antigen may be peptides from an adenovirus, retrovirus,picornavirus, herpesvirus, rotaviruses, hantaviruses, coronavirus,togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus,bunyavirus, arenavirus, reovirus, papilomavirus, parvovirus, poxvirus,hepadnavirus, or spongiform virus. In certain specific, non-limitingexamples, the at least one viral antigen are peptides obtained from atleast one of HIV, CMV, hepatitis A, B, and C, influenza, measles, polio,smallpox, rubella; respiratory syncytial, herpes simplex, varicellazoster, Epstein-Barr, Japanese encephalitis, rabies, flu, and/or coldviruses.

In one aspect, the one or more of the antigenic peptides are selectedfrom at least one of: Nef (66-97): VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL (SEQID NO.: 120); Nef (116-145): HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL (SEQ ID NO.:121); Gag p17 (17-35): EKIRLRPGGKKKYKLKHIV (SEQ ID NO.: 122); Gagp17-p24 (253-284): NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD (SEQ ID NO.: 123);or Pol 325-355 (RT 158-188) is: AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY (SEQ IDNO.: 124). In one aspect, the fusion protein peptides are separated byone or more linkers selected from: SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ IDNO.: 23); PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 24);TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 25); or TNGSITVAATAPTVTPTVNATPSAA(SEQ ID NO.: 26).

Antigenic targets that may be delivered using the anti-CD40-antigenvaccines of the present invention include genes encoding antigens suchas viral antigens, bacterial antigens, fungal antigens or parasiticantigens. Pathogens include trypanosomes, tapeworms, roundworms,helminthes, malaria. Tumor markers, such as fetal antigen or prostatespecific antigen, may be targeted in this manner. Other examplesinclude: HIV env proteins and hepatitis B surface antigen.Administration of a vector according to the present invention forvaccination purposes would require that the vector-associated antigensbe sufficiently non-immunogenic to enable long-term expression of thetransgene, for which a strong immune response would be desired. In somecases, vaccination of an individual may only be required infrequently,such as yearly or biennially, and provide long-term immunologicprotection against the infectious agent. Specific examples of organisms,allergens and nucleic and amino sequences for use in vectors andultimately as antigens with the present invention may be found in U.S.Pat. No. 6,541,011, relevant portions incorporated herein by reference,in particular, the tables that match organisms and specific sequencesthat may be used with the present invention.

Bacterial antigens for use with the anti-CD40-antigen vaccines disclosedherein include, but are not limited to, e.g., bacterial antigens such aspertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3,adenylate cyclase and other pertussis bacterial antigen components;diptheria bacterial antigens such as diptheria toxin or toxoid and otherdiptheria bacterial antigen components; tetanus bacterial antigens suchas tetanus toxin or toxoid and other tetanus bacterial antigencomponents; streptococcal bacterial antigens such as M proteins andother streptococcal bacterial antigen components; gram-negative bacillibacterial antigens such as lipopolysaccharides and other gram-negativebacterial antigen components, Mycobacterium tuberculosis bacterialantigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDamajor secreted protein, antigen 85A and other mycobacterial antigencomponents; Helicobacter pylori bacterial antigen components;pneumococcal bacterial antigens such as pneumolysin, pneumococcalcapsular polysaccharides and other pneumococcal bacterial antigencomponents; haemophilus influenza bacterial antigens such as capsularpolysaccharides and other haemophilus influenza bacterial antigencomponents; anthrax bacterial antigens such as anthrax protectiveantigen and other anthrax bacterial antigen components; rickettsiaebacterial antigens such as rompA and other rickettsiae bacterial antigencomponent. Also included with the bacterial antigens described hereinare any other bacterial, mycobacterial, mycoplasmal, rickettsial, orchlamydial antigens. Partial or whole pathogens may also be: haemophilusinfluenza; Plasmodium falciparum; neisseria meningitidis; streptococcuspneumoniae; neisseria gonorrhoeae; salmonella serotype typhi; shigella;vibrio cholerae; Dengue Fever; Encephalitides; Japanese Encephalitis;lyme disease; Yersinia pestis; west nile virus; yellow fever; tularemia;hepatitis (viral; bacterial); RSV (respiratory syncytial virus); HPIV 1and HPIV 3; adenovirus; small pox; allergies and cancers.

Fungal antigens for use with compositions and methods of the inventioninclude, but are not limited to, e.g., candida fungal antigencomponents; histoplasma fungal antigens such as heat shock protein 60(HSP60) and other histoplasma fungal antigen components; cryptococcalfungal antigens such as capsular polysaccharides and other cryptococcalfungal antigen components; coccidiodes fungal antigens such as spheruleantigens and other coccidiodes fungal antigen components; and tineafungal antigens such as trichophytin and other coccidiodes fungalantigen components.

Examples of protozoal and other parasitic antigens include, but are notlimited to, e.g., plasmodium falciparum antigens such as merozoitesurface antigens, sporozoite surface antigens, circumsporozoiteantigens, gametocyte/gamete surface antigens, blood-stage antigen pf155/RESA and other plasmodial antigen components; toxoplasma antigenssuch as SAG-1, p30 and other toxoplasmal antigen components;schistosomae antigens such as glutathione-S-transferase, paramyosin, andother schistosomal antigen components; leishmania major and otherleishmaniae antigens such as gp63, lipophosphoglycan and its associatedprotein and other leishmanial antigen components; and trypanosoma cruziantigens such as the 75-77 kDa antigen, the 56 kDa antigen and othertrypanosomal antigen components.

Antigen that can be targeted using the anti-CD40-antigen vaccines of thepresent invention will generally be selected based on a number offactors, including: likelihood of internalization, level of immune cellspecificity, type of immune cell targeted, level of immune cell maturityand/or activation and the like. In this embodiment, the antibodies maybe mono- or bi-specific antibodies that include one anti-CD40 bindingdomain and one binding domain against a second antigen, e.g., cellsurface markers for dendritic cells such as, MHC class I, MHC Class II,B7-2, CD18, CD29, CD31, CD43, CD44, CD45, CD54, CD58, CD83, CD86,CMRF-44, CMRF-56, DCIR and/or Dectin-1 and the like; while in some casesalso having the absence of CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD 19,CD20, CD56, and/or CD57. Examples of cell surface markers for antigenpresenting cells include, but are not limited to, MHC class I, MHC ClassII, CD45, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1 and/orFcγ receptor. Examples of cell surface markers for T cells include, butare not limited to, CD3, CD4, CD8, CD 14, CD20, CD11b, CD16, CD45 andHLA-DR.

Target antigens on cell surfaces for delivery include thosecharacteristic of tumor antigens typically derived from the cellsurface, cytoplasm, nucleus, organelles and the like of cells of tumortissue. Examples of tumor targets for the antibody portion of thepresent invention include, without limitation, hematological cancerssuch as leukemias and lymphomas, neurological tumors such asastrocytomas or glioblastomas, melanoma, breast cancer, lung cancer,head and neck cancer, gastrointestinal tumors such as gastric or coloncancer, liver cancer, pancreatic cancer, genitourinary tumors suchcervix, uterus, ovarian cancer, vaginal cancer, testicular cancer,prostate cancer or penile cancer, bone tumors, vascular tumors, orcancers of the lip, nasopharynx, pharynx and oral cavity, esophagus,rectum, gall bladder, biliary tree, larynx, lung and bronchus, bladder,kidney, brain and other parts of the nervous system, thyroid, Hodgkin'sdisease, non-Hodgkin's lymphoma, multiple myeloma and leukemia.

Examples of antigens that may be delivered alone or in combination toimmune cells for antigen presentation using the present inventionincludes tumor proteins, e.g., mutated oncogenes; viral proteinsassociated with tumors; and tumor mucins and glycolipids. The antigensmay be viral proteins associated with tumors would be those from theclasses of viruses noted above. Certain antigens may be characteristicof tumors (one subset being proteins not usually expressed by a tumorprecursor cell), or may be a protein which is normally expressed in atumor precursor cell, but having a mutation characteristic of a tumor.Other antigens include mutant variant(s) of the normal protein having analtered activity or subcellular distribution, e.g., mutations of genesgiving rise to tumor antigens.

Specific non-limiting examples of tumor antigens for use in ananti-CD40-fusion protein vaccine include, e.g., CEA, prostate specificantigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC (Mucin)(e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc,tyrosinase, MART (melanoma antigen), Pmel 17(gp100), GnT-V intron Vsequence (N-acetylglucoaminyltransferase V intron V sequence), ProstateCa psm, PRAME (melanoma antigen), β-catenin, MUM-1-B (melanomaubiquitous mutated gene product), GAGE (melanoma antigen) 1, MAGE, BAGE(melanoma antigen) 2-10, c-ERB2 (Her2/neu), DAGE, EBNA (Epstein-BanVirus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7,p53, lung resistance protein (LRP), Bcl-2, Ki-67, Cyclin B1, gp100,Survivin, and NYESO-1

In addition, the immunogenic molecule can be an autoantigen involved inthe initiation and/or propagation of an autoimmune disease, thepathology of which is largely due to the activity of antibodies specificfor a molecule expressed by the relevant target organ, tissue, or cells,e.g., SLE or MG. In such diseases, it can be desirable to direct anongoing antibody-mediated (i.e., a Th2-type) immune response to therelevant autoantigen towards a cellular (i.e., a Th1-type) immuneresponse. Alternatively, it can be desirable to prevent onset of ordecrease the level of a Th2 response to the autoantigen in a subject nothaving, but who is suspected of being susceptible to, the relevantautoimmune disease by prophylactically inducing a Th1 response to theappropriate autoantigen. Autoantigens of interest include, withoutlimitation: (a) with respect to SLE, the Smith protein, RNPribonucleoprotein, and the SS-A and SS-B proteins; and (b) with respectto MG, the acetylcholine receptor. Examples of other miscellaneousantigens involved in one or more types of autoimmune response include,e.g., endogenous hormones such as luteinizing hormone, follicularstimulating hormone, testosterone, growth hormone, prolactin, and otherhormones.

Antigens involved in autoimmune diseases, allergy, and graft rejectioncan be used in the compositions and methods of the invention. Forexample, an antigen involved in any one or more of the followingautoimmune diseases or disorders can be used in the present invention:diabetes, diabetes mellitus, arthritis (including rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis),multiple sclerosis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, includingkeratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopeciaareata, allergic responses due to arthropod bite reactions, Crohn'sdisease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.Examples of antigens involved in autoimmune disease include glutamicacid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelinproteolipid protein, acetylcholine receptor components, thyroglobulin,and the thyroid stimulating hormone (TSH) receptor.

Examples of antigens involved in allergy include pollen antigens such asJapanese cedar pollen antigens, ragweed pollen antigens, rye grasspollen antigens, animal derived antigens such as dust mite antigens andfeline antigens, histocompatiblity antigens, and penicillin and othertherapeutic drugs. Examples of antigens involved in graft rejectioninclude antigenic components of the graft to be transplanted into thegraft recipient such as heart, lung, liver, pancreas, kidney, and neuralgraft components. The antigen may be an altered peptide ligand useful intreating an autoimmune disease.

It will be appreciated by those of skill in the art that the sequence ofany protein effector molecule may be altered in a manner that does notsubstantially affect the functional advantages of the effector protein.For example, glycine and alanine are typically considered to beinterchangeable as are aspartic acid and glutamic acid and asparagineand glutamine One of skill in the art will recognize that many differentvariations of effector sequences will encode effectors with roughly thesame activity as the native effector. The effector molecule and theantibody may be conjugated by chemical or by recombinant means asdescribed above. Chemical modifications include, for example,derivitization for the purpose of linking the effector molecule and theantibody to each other, either directly or through a linking compound,by methods that are well known in the art of protein chemistry. Bothcovalent and noncovalent attachment means may be used with the humanizedantibodies of the present invention.

The procedure for attaching an effector molecule to an antibody willvary according to the chemical structure of the moiety to be attached tothe antibody. Polypeptides typically contain a variety of functionalgroups; e.g., carboxylic acid (COOH), free amine (—NH₂) or sulfhydryl(—SH) groups, which are available for reaction with a suitablefunctional group on an antibody to result in the binding of the effectormolecule. Alternatively, the antibody can be derivatized to expose or toattach additional reactive functional groups, e.g., by attachment of anyof a number of linker molecules such as those available from PierceChemical Company, Rockford Ill.

The linker is capable of forming covalent bonds to both the antibody andto the effector molecule. Suitable linkers are well known to those ofskill in the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the antibody and the effector molecule are polypeptides,the linkers may be joined to the constituent amino acids through theirside groups (e.g., through a disulfide linkage to cysteine). However, ina preferred embodiment, the linkers will be joined to the alpha carbonamino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerthat is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used.

Exemplary chemical modifications of the effector molecule and theantibody of the present invention also include derivitization withpolyethylene glycol (PEG) to extend time of residence in the circulatorysystem and reduce immunogenicity, according to well known methods (Seefor example, Lisi, et al., Applied Biochem. 4:19 (1982); Beauchamp, etal., Anal Biochem. 131:25 (1982); and Goodson, et al., Bio/Technology8:343 (1990)).

The present invention contemplates vaccines for use in both active andpassive immunization embodiments. Immunogenic compositions, proposed tobe suitable for use as a vaccine, may be prepared most readily directlyfrom immunogenic T-cell stimulating peptides prepared in a mannerdisclosed herein. The final vaccination material is dialyzed extensivelyto remove undesired small molecular weight molecules and/or lyophilizedfor more ready formulation into a desired vehicle. In certain embodimentof the present invention, the compositions and methods of the presentinvention are used to manufacture a cellular vaccine, e.g., theantigen-delivering anti-CD40 binding portion of the antibody is used todirect the antigen(s) to an antigen presenting cell, which then “loads”the antigen onto MHC proteins for presentation. The cellular vaccine is,therefore, the antigen presenting cell that has been loaded using thecompositions of the present invention to generate antigen-loaded antigenpresenting cells.

When the vaccine is the anti-CD40 binding protein itself, e.g., acomplete antibody or fragments thereof, then these “active ingredients”can be made into vaccines using methods understood in the art, e.g.,U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; and4,578,770, relevant portions incorporated herein by reference.Typically, such vaccines are prepared as injectables, e.g., as liquidsolutions or suspensions or solid forms suitable for re-suspension inliquid prior to injection. The preparation may also be emulsified. Theactive immunogenic ingredient is often mixed with excipients, which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants, whichenhance the effectiveness of the vaccines.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to generate an immune response. Precise amounts of cells oractive ingredient required to be administered depend on the judgment ofthe practitioner. However, suitable dosage ranges are of the order of afew thousand cells (to millions of cells) for cellular vaccines. Forstandard epitope or epitope delivery vaccines then the vaccine may beseveral hundred micrograms active ingredient per vaccination. Suitableregimes for initial administration and booster shots are also variable,but are typified by an initial administration followed by subsequentinoculations or other administrations.

The manner of application may vary widely, however, certain embodimentsherein will most likely be delivered intravenously or at the site of atumor or infection directly. Regardless, any of the conventional methodsfor administration of a vaccine are applicable. The dosage of thevaccine will depend on the route of administration and will varyaccording to the size of the host.

In many instances, it will be desirable to have multiple administrationsof the vaccine, e.g., four to six vaccinations provided weekly or everyother week. A normal vaccination regimen will often occur in two totwelve week intervals or from three to six week intervals. Periodicboosters at intervals of 1-5 years, usually three years, may bedesirable to maintain protective levels of the immune response or upon alikelihood of a remission or re-infection. The course of theimmunization may be followed by assays for, e.g., T cell activation,cytokine secretion or even antibody production, most commonly conductedin vitro. These immune response assays are well known and may be foundin a wide variety of patents and as taught herein.

A vaccine of the present invention may be provided in one or more “unitdoses” depending on whether the nucleic acid vectors are used, the finalpurified proteins, or the final vaccine form is used. Unit dose isdefined as containing a predetermined-quantity of the therapeuticcomposition calculated to produce the desired responses in associationwith its administration, i.e., the appropriate route and treatmentregimen. The quantity to be administered, and the particular route andformulation, are within the skill of those in the clinical arts. Thesubject to be treated may also be evaluated, in particular, the state ofthe subject's immune system and the protection desired. A unit dose neednot be administered as a single injection but may include continuousinfusion over a set period of time. Unit dose of the present inventionmay conveniently be described in terms of DNA/kg (or protein/Kg) bodyweight, with ranges between about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1,10, 50, 100, 1,000 or more mg/DNA or protein/kg body weight areadministered.

Likewise, the amount of anti-CD40-antigen vaccine delivered can varyfrom about 0.2 to about 8.0 mg/kg body weight. Thus, in particularembodiments, 0.4 mg, 0.5 mg, 0.8 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0mg, 4.0 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg and 7.5 mg of thevaccine may be delivered to an individual in vivo. The dosage of vaccineto be administered depends to a great extent on the weight and physicalcondition of the subject being treated as well as the route ofadministration and the frequency of treatment. A pharmaceuticalcomposition that includes a naked polynucleotide prebound to a liposomalor viral delivery vector may be administered in amounts ranging from 1μg to 1 mg polynucleotide to 1 μg to 100 mg protein. Thus, particularcompositions may include between about 1 μg, 5 μg, 10 μg, 20 μg, 30 μg,40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500μg, 600 μg, 700 μg, 800 μg, 900 μg or 1,000 μg polynucleotide or proteinthat is bound independently to 1 μg, 5 μg, 10 μg, 20 μg, 3.0 μg, 40 μg50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500 μg, 600μg, 700 μg, 800 μg, 900 μg, 1 mg, 1.5 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg vector.

Antibodies of the present invention may optionally be covalently ornon-covalently linked to a detectable label. Detectable labels suitablefor such use include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical methods. Useful labels in the present invention includemagnetic beads (e.g. DYNABEADS), fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

Methods of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

The antibody and/or immunoconjugate compositions of this invention areparticularly useful for parenteral administration, such as intravenousadministration or administration into a body cavity. The compositionsfor administration will commonly comprise a solution of the antibodyand/or immunoconjugate dissolved in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., buffered saline and the like. These solutions aresterile and generally free of undesirable matter. These compositions maybe sterilized by conventional, well-known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration offusion protein in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs.

Thus, a typical pharmaceutical immunoconjugate composition of thepresent invention for intravenous administration would be about 0.1 to10 mg per patient per day. Dosages from 0.1 up to about 100 mg perpatient per day may be used. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as REMINGTON'SPHARMACEUTICAL SCIENCE, 19TH ED., Mack Publishing Company, Easton, Pa.(1995).

The compositions of the present invention can be administered fortherapeutic treatments. In therapeutic applications, compositions areadministered to a patient suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. An effective amount of the compound is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the proteins of this invention to effectively treat thepatient. Preferably, the dosage is administered once but may be appliedperiodically until either a therapeutic result is achieved or until sideeffects warrant discontinuation of therapy. Generally, the dose issufficient to treat or ameliorate symptoms or signs of disease withoutproducing unacceptable toxicity to the patient.

Controlled release parenteral formulations of the immunoconjugatecompositions of the present invention can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., THERAPEUTIC PEPTIDES AND PROTEINS:FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein as a central core. In microspheres the therapeuticis dispersed throughout the particle. Particles, microspheres, andmicrocapsules smaller than about 1 μm are generally referred to asnanoparticles, nanospheres, and nanocapsules, respectively. Capillarieshave a diameter of approximately 5 μm so that only nanoparticles areadministered intravenously. Microparticles are typically around 100 μmin diameter and are administered subcutaneously or intramuscularly.

Polymers can be used for ion-controlled release of immunoconjugatecompositions of the present invention. Various degradable andnon-degradable polymeric matrices for use in controlled drug deliveryare known in the art (Langer, R., Accounts Chem. Res. 26:537-542(1993)). For example, the block copolymer, poloxamer 407® exists as aviscous yet mobile liquid at low temperatures but forms a semisolid gelat body temperature, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins, and/or liposomes may be used forcontrolled release as well as drug targeting of the lipid-capsulateddrug. Numerous additional systems for controlled delivery of therapeuticproteins are known. See, e.g., U.S. Pat. Nos. 5,055,303, 5,188,837,4,235,871, 4,501,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303;5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206,5,271,961; 5,254,342 and 5,534,496, relevant portions of each of whichare incorporated herein by reference.

Among various uses of the antibodies of the invention are included avariety of disease conditions caused by specific human cells. Forexample, for a humanized version of the mouse anti-CD40_12E12.3F3 (ATCCAccession No. PTA-9854), anti-CD40_12B4.2C10 (Deposit No. HS446, ATCCAccession No. PTA-10653), and anti-CD40_11B6.1C3 (Deposit No. HS440,ATCC Accession No. PTA-10652), antibodies disclosed herein, oneapplication for antibodies is the treatment, contacting, imaging,activation or deactivation of cells expressing CD40.

In another embodiment, this invention provides kits for the delivery ofantigens, e.g., CD40 or an immunoreactive fragment thereof, conjugatedor in the form of a fusion protein with one or more T cell or B cellepitopes. A “biological sample” as used herein is a sample of biologicaltissue or fluid that contains the antigen. Such samples include, but arenot limited to, tissue from biopsy, blood, and blood cells (e.g., whitecells). Preferably, the cells are lymphocytes, e.g., dendritic cells.Biological samples also include sections of tissues, such as frozensections taken for histological purposes. A biological sample istypically obtained from a multicellular eukaryote, preferably a mammalsuch as rat, mouse, cow, dog, guinea pig, or rabbit, and more preferablya primate, such as a macaque, chimpanzee, or human. Most preferably, thesample is from a human. The antibodies of the invention may also be usedin vivo, for example, as a diagnostic tool for in vivo imaging.

Kits will typically comprise a nucleic acid sequence that encodes anantibody of the present invention (or fragment thereof) with one or moreframework portions or multiple cloning sites at the carboxy-terminal endinto which the coding sequences for one or more antigens may beinserted. In some embodiments, the antibody will be a humanizedanti-CD40 Fv fragment, such as an scFv or dsFv fragment. In addition thekits will typically include instructional materials disclosing methodsof use of an antibody of the present invention (e.g. for loading intodendritic cells prior to immunization with the dendritic cells, whichcan be autologous dendritic cells). The kits may also include additionalcomponents to facilitate the particular application for which the kit isdesigned. Thus, for example, the kit may additionally contain methods ofdetecting the label (e.g. enzyme substrates for enzymatic labels, filtersets to detect fluorescent labels, appropriate secondary labels such asa sheep anti-mouse-HRP, or the like). The kits may additionally includebuffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

In another set of uses for the invention, antibodies targeted byantibodies of the invention can be used to purge targeted cells from apopulation of cells in a culture. For example, if a specific populationof T cells is preferred, the antibodies of the present invention may beused to enrich a population of T cells having the opposite effect of theon-going immune response. Thus, for example, cells cultured from apatient having a cancer can be purged of cancer cells by providing thepatient with dendritic cells that were antigen loaded using theantibodies of the invention as a targeting moiety for the antigens thatwill trigger an immune response against the cancer, virus or otherpathogen. Likewise, the antibodies can be used to increase thepopulation of regulatory T cells or drive the immune response toward oraway from a cytotoxic T cell response or even drive a B cell response.

anti-CD40_12E12.3F3 anti-CD40_12E12.3F3_H-V-hIgG4H-C - underlinedregion shows the Heavy chain V region amino acid sequence:(SEQ ID NO.: 1) MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASanti-CD40_12E12.3F3_K-V-hIgGK-C - underlinedregion shows the Light chain V region amino acid sequence(SEQ ID NO.: 2) MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTIGNLEPEDIATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC- anti-CD40_12B4.2C10anti-CD40_12B4.2C10 Heavy Chain: (SEQ ID No.: 3)MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYVLHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGYPAYSGYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSS GVHTFPAVLQKGEFVanti-CD40_12B4.2C10 Light Chain: (SEQ ID No.: 4)MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCHHGNTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECanti-CD40_12B4.2C10 Light Chain - alternative clone (17K6)(SEQ ID No.: 5) MDFQVQIFSFLLISASVIMSRGQIVLTQSPAILSASPGEKVTMTCSASSSVSYMYRYQQKPGSSPKPWIYGTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC anti-CD40_11B6.1C3anti-CD40_11B6.1C3 Heavy Chain: (SEQ ID No.: 6)MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATSYNQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCAREDYVYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPA VLQKGEFVanti-CD40_11B6.1C3 Light Chain: (SEQ ID No.: 7)MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFALKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNR NEC[anti-CD40_12E12.3F3_H-V-hIgG4H-C] - underlinedregion shows the Heavy chain V region sequence: (SEQ ID NO.: 8)ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCTGA[anti-CD40_12E12.3F3_K-V-hIgGK-C] - underlinedregion shows the Light chain V region sequence (SEQ ID NO.: 9)ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTAGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCAATTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCGGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTTTAATAAGCTTCCTCCGACGTTCGGTGGAGGCACCAAACTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGanti-CD40_12B4.2C10_H-V-hIgG4H-C heavy chain (SEQ ID NO.: 10)ATGGAATGGAGTTGGATATTTCTCTTTCTTCTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTATGTTTTGCACTGGGTGAAACAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGGGGCTATCCGGCCTACTCTGGGTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCTGAanti-CD40_12B4.2C10_K-V-hIgGK-C (variant 1) light chain (SEQ ID NO.: 11)ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCCAGGGGACAAATTGTTCTCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACAGGTACCAGCAGAAGCCAGGATCCTCACCCAAACCCTGGATTTATGGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAATATCATAGTTACCCGCTCACGTTCGGTGCTGGGACCAAGCTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TAGanti-CD40_12B4.2C10_K-V-hIgGK-C (Variant 2) light chain (SEQ ID NO.: 12)ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCATCATGGTAATACGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGanti-CD40_11B6.1C3_H-V-hIgG4H-C heavy chain (SEQ ID NO.: 14)ATGGGATGGAGCTGGATCTTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCTCTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATGCACTGGGTGAAGCAAAGCCATGTAAAGAGCCTTGAGTGGATTGGACGTATTAATCCTTACAATGGTGCTACTAGCTACAACCAGAATTTCAAGGACAAGGCCAGCTTGACTGTAGATAAGTCCTCCAGCACAGCCTACATGGAGCTCCACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGAGGACTACGTCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCTGGGCTGCCTCGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCTGA anti-CD40_11B6.1C3_K-V-hIgGK-C light chain(SEQ ID NO: 15) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCGCACTCAAGATCAGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTAG

EXAMPLE 1 Anti-CD40-HIV Peptides Vaccine

Five 19- to 32-amino-acid long sequences were selected from amultiplicity of cytotoxic T lymphocyte (CTL) epitopes identified in theHIV-1 Nef, Gag and Env proteins in the context of different MHC-class Imolecules. It has been reported that CTL responses can be inducedefficiently by lipopeptide vaccines in mice, in primates, and in humans.The five HIV peptides were then modified in C-terminal position by a(Palm)-NH2 group and the five HIV peptide sequences have been welldescribed in the scientific literature [e.g., Characterization of amulti-lipopeptides mixture used as an HIV-1 vaccine candidate (1999)Klinguer et al., Vaccine, Volume 18, 259-267] and in a patentapplication [Cytotoxic T lymphocyte-inducing lipopeptides and use asvaccines. Gras-Masse H. et al., Patent No. EPO491628 (1992 Jun. 24);U.S. Pat. No. 5,871,746 (1999 Feb. 16)].

A very desirable HIV vaccine would be composed of recombinantanti-dendritic cell receptor antibody fused to the above HIV peptides.The present invention includes compositions and methods to efficientlyproduce proteins and HIV vaccines.

The sequences shown below are the amino-acid sequences of the fiveselected HIV peptides and the amino-acid positions within each HIVprotein are in brackets.

Nef (66-97) is: VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL (SEQ ID NO.: 16)Nef (116-145) is: HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL (SEQ ID NO.: 17)Gag p17 (17-35) is: EKIRLRPGGKKKYKLKHIV (SEQ ID NO.: 18)Gag p17-p24 (253-284) is: NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD(SEQ ID NO.: 19) Pol 325-355 (RT 158-188) is:AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY (SEQ ID NO.: 20)

The present invention includes compositions and methods for assemblingconstructs encoding HIV peptides and Flexible linker sequences. TheHeavy chain expression vectors typically have a Nhe I site [g|ctagc]appended to the Heavy chain C-terminal residue codon, or [for flex-v1vectors] to the C-terminal codon of the flex-v1 sequence. Flexiblelinker sequences or HIV peptide sequences have an Spe I site [a|tagt]preceding the N-terminal flexible linker or HIV peptide codon, a Nhe Isite appended to the C-terminal flexible linker or HIV peptide codon,followed by a TGA stop codon, followed by a Eco RI site, followed by aNot I site. Such flexible linker or HIV peptide Spe I-Not I fragmentsare inserted into the Heavy chain vector prepared with Nhe I-Not Idigestion. Nhe I and Spe I are compatible sites, but when ligated[g|ctagt] is no longer either a Nhe I or Spe I site. Thus additional SpeI-Not I flexible linker or HIV peptide fragments can be inserted intothe new Nhe I-Not I interval distal to the initial flexible linker orHIV peptide. In this way, strings of HIV peptide and/or flexible linkercoding regions can be appended to the expression vector Heavy chaincoding region.

FIG. 1 shows protein A affinity recombinant antibodies fused to variousHIV peptides (lanes 1 to 5) secreted from transfected 293F cells,analyzed by reducing SDS-PAGE and Coomassie Brilliant Blue staining FIG.2 shows protein A affinity purified recombinant antibodies fused tovarious HIV peptides (Lanes 1 and 2) secreted from transfected 293Fcells, then analyzed by reducing SDS-PAGE and Coomassie Brilliant Bluestaining. FIG. 3 shows protein A affinity purified recombinantantibodies fused to various HIV peptide strings (Lanes 1 to 5) secretedfrom transfected 293F cells, then analyzed by reducing SDS.PAGE andCoomassie Brilliant Blue staining. FIG. 4 shows protein A affinitypurified recombinant antibodies fused to various HIV peptide strings(Lanes 1 to 6) secreted from transfected 293F cells, then analyzed byreducing SDS.PAGE and Coomassie Brilliant Blue staining.

EXAMPLE 2 HIV Peptides Vaccine—In Vitro Antigen-Targeting Biology

Anti-CD40.LIPO5 HIV peptides vaccine tests on HIV patients in vitro. Tostudy the ability of αCD40.LIPO5 HIV peptide fusion recombinant antibody(αCD40.LIPO5 rAb) to mediate antigen presentation, the fusion rAb wasadded to blood cells from HIV-infected individuals and measured cytokineproduction form peripheral blood mononuclear cells (PBMCs).

FIG. 5 describes the protocol used in vitro to assay the potency ofαCD40.LIPO5 HIV peptide fusion recombinant antibody (αCD40.LIPO5 rAb) toelicit the expansion of antigen-specific T cells in the context of aPBMC culture. Briefly, PBMCs (2×10⁶ cells/ml) from apheresis of HIVpatients are incubated with a dose range of αCD40.LIPO5 HIV peptidevaccine. On day 2, 100 U/ml IL-2 are added to the culture and then, themedia is refreshed every 2 days with 100 U/ml IL-2. On day 10, theexpanded cells are challenged for 48 h with the individual long peptidescorresponding to the 5 HIV peptide sequences incorporated in theαCD40.LIPO5 HIV peptide fusion rAb. Then, culture supernatants areharvested and assessed for cytokine production (by the T cells with Tcell receptor [TCR] specificities for peptide sequences) using multiplexbeads assay (Luminex). Antigen-specific cytokine production detected insuch an assay, if it depends on the presence of the anti-CD40.LIPO5 HIVpeptide vaccine, reflects vaccine uptake by antigen presenting cells[APC] in the culture, and processing [proteolytic degradation] andpresentation of peptides on MHC. The antigen-MHC complexes arerecognized by T cells with TCR that recognize only the particular HIVantigen-MHC complex. In a HIV patient, such cells are likely to bememory T cells that expanded in the patient in response to the HIVinfection.

Epitopes from all 5 HIV peptide regions of the vaccine can be presentedby APCs. The scheme in FIG. 5 was used to assay the in vitro expansionof HIV peptide-specific T cells in response to anti-CD40.LIPO5 peptidevaccine. Results from 7 individuals are shown in FIG. 6 and indicatethat the αCD40.LIPO5 HIV peptide fusion rAb elicited HIVpeptide-specific IFNγ responses in all of the patients studied. Thus,the α-CD40.LIPO5 HIV peptide fusion rAb allows DCs to cross present atleast 1 or 2 different peptides out of the 5 peptides within the vaccineto the T cells of each individual. However, the set of HIV peptides thatstimulated IFNγ production was different for each patient—most likelyreflecting different pools of memory T cells for HIV specificity.

FIG. 6 shows the HIV peptide-specific IFN-γ production in PBMCs from HIVpatients incubated with various concentrations of anti-CD40.LIPO5peptide string vaccine. C is the control group, which received novaccine, and defines the baseline response of the culture to eachpeptide.

FIG. 7 is a summary of αCD40.LIPO5 peptide vaccine responses against the5 peptide regions from 8 HIV patients. The data are based onpeptide-specific IFN-γ production. FIG. 7 shows that theantigen-specific responses observed in 8 HIV patients. The datademonstrate that all HIV peptide regions on the vaccine have thecapacity to be processed and presented to T cells—assuming the likelysituation that responses to these peptides will only be observed if theappropriate TCR-bearing cells are present. Thus, each patient has acharacteristic spectrum of such cells.

The αCD40.LIPO5 peptide vaccine can evoke the proliferation ofantigen-specific T cells capable of secreting a wide spectrum ofcytokines

FIG. 8A-B shows that αCD40.LIPO5 HIV peptide vaccine elicits expansionof HIV peptide-specific T cells capable of secreting multiplecytokines—a desirable feature in a vaccine. In FIG. 8A-B αCD40.LIPO5 HIVpeptide vaccine elicits gag253, nef66, nef116 and pol325peptide-specific responses characterized by production of multiplecytokines. This is patient A5.

Anti-CD40.LIPO5 HIV Peptide Vaccination of Ex Vivo DCs.

FIG. 9 shows the protocol for testing αCD40.LIPO5 HIV peptide vaccinefor its ability to direct the expansion of antigen-specific T cellsresulting from targeted uptake by DCs and presentation of peptideepitopes on their surface MHC complex. Briefly, HIV patient monocytesare differentiated into DCs by culture for 2 days with IFNα and GM-CSF.Different doses αCD40.LIPO5 HIV peptide vaccine or a mix of the 5peptides are then added for 18 h. Autologous T cells were added to theco-culture (at a ratio of 1:20) on day 3. On day 5, 100 U/ml IL-2 areadded to the culture and then, the media is refreshed every 2 days with100 U/ml IL-2. On day 10, the expanded cells are rechallenged for 48 hwith the individual long peptides corresponding to the 5 HIV peptidesequences incorporated in the αCD40.LIPO5 HIV peptide fusion rAb. Then,culture supernatants are harvested and assessed for cytokine productionusing Luminex.

FIG. 10A-B shows the cytokine secretion in response to HIV peptides fromDC-T cell co-cultures treated with various doses of αCD40.LIPO5 HIVpeptide vaccine. This is patient A10. The results in the patient A10shown in FIG. 10A-B demonstrate expansion of antigen-specific T cellscorresponding to epitopes within the gag17, gag253, and pol325 HIVpeptide regions. In most instances, there is concordance of responsesbetween αCD40.LIPO5 HIV peptide vaccine and non-LIPO5 vaccine [mixtureof 5 non-lipidated HIV peptides with sequences corresponding to those inthe αCD40.LIPO5 HIV peptide vaccine]. Thus, the αCD40.LIPO5 HIV peptidevaccine functions well in this in vitro setting where cultured DCseffectively process and present the HIV antigens to T cells. Thisexemplifies use of the αCD40.LIPO5 HIV peptide vaccine for ex vivovaccination, whereby the ‘vaccinated DCs’ would be cryopreserved forfuture re-injection into the same patient.

αCD40.LIPO5 HIV peptide vaccine—possible immune effect of the flexiblelinker regions. It is possible that the flexible linker sequencesinterspersing the HIV peptide sequences within the αCD40.LIPO5 HIVpeptide vaccine themselves contain T cell epitopes. FIG. 11A-B showsthat patient A4 does not appear to have a significant pool of memory Tcells with specificities to the five flexible linker sequences withinαCD40.LIPO5 HIV peptide vaccine. In FIG. 11A-B, PBMCs from patient A4treated with the αCD40.LIPO5 HIV peptide vaccine elicit expansion ofantigen-specific T cells with specificity to the gag253 region, but notto the flexible linker sequences. The protocol describe in FIG. 9 wasused, with the flexible linker long peptides corresponding in sequenceto the bold areas, the HIV peptides are in bold-italics, shown in thesequence below.

αCD40.LIPO5 HIV peptide vaccine heavy chain sequence showing flexiblelinker regions in bold, joining sequences underlined and HIV peptideregions shaded in bold italics.

(SEQ ID NO.: 21) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GKASQTPTNTISVTPTNNSTPTNNSNPKPNP AS

AS SSVSPTTSVHPTPTSVPPTPTKSSP AS

AS PTSTPADSSTITPTATPTATPTIKG AS

AS TVTPTATATPSAIVTTITPTATTKP A S

AS TNGSITVAATAPTVT PTVNATPSAA AS

AS.

In FIG. 12A, the PBMCs from patient A3 treated with the αCD40.LIPO5 HIVpeptide vaccine elicit expansion of antigen-specific T cells withspecificities to the gag253, nef66, and nef116 regions, but not to theflexible linker sequences. The protocol described in FIG. 1 was used,with the flexible linker long peptides corresponding in sequence to thebold areas shown in FIG. 8A-B.

FIGS. 12B-1 and 12B-2 shows HIV antigen-specific T cell responses evokedfrom HIV patient A17 PBMCs incubated with 30 nM of three different HIV5peptide DC targeting vaccines. Cells were cultured for 10 days with IL-2and then stimulated with individual long peptides corresponding to the 5HIV peptide sequences encompassed within the DC-targeting vaccines.After 1 hr brefeldin A was added and incubation continued for a further5 hrs before staining for FACS analysis. The FACS plots show IFNg andCD8 staining on CD3+ T cells. Circles indicate significantvaccine-evoked expansion of IFNg+ cells compared to cells from PBMCscultured without vaccine. CD8− cells are CD4+ T cells. The data showthat that anti-CD40.HIV5pep vaccine evokes a strong expansion of nef66(N66)-specific CD8+ T cells which is not seen with the other DCtargeting vehicles.

These are data based on the LIPO5 HIV peptide string. For example theanti-CD40 Heavy chain isanti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-Pep-gag17-f1-gag253-f2-nef116-f3-nef66-f4-pol158]with sequence:

(SEQ ID NO.: 22) EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASEKIRLRPGGKKKYKLKHIVASSSVSPTTSVHPTPTSVPPTPTKSSPASNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDASPTSTPADSSTITPTATPTATPTIKGASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLASTVTPTATATPSAIVTTITPTATTKPASVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLASTNGSITVAATAPTVTPTVNATPSAAASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYAS.

FIGS. 12C-1 and 12C-2 is a similar study to that show in FIGS. 12B-1 and12B-2, except that the PBMCs are from a different HIV patient (A2). Thedata show antigen-specific CD4+ and CD8+ T cell responses evoked byanti-CD40.HIV5pep but not the other DC-targeting vaccines, or by amixture of the peptides themselves.

FIG. 12D shows that, based on analysis of 15 different HIV peptideresponses [5 peptide regions sampled in 3 patients], anti-CD40.HIV5pepvaccine is clearly superior to anti-DCIR.HIV5pep, anti-LOX-1.HIV5pep andnon-LIPO5 mix for eliciting a broad range of HIV peptide-specific CD8+and CD4+ T responses.

The immunogenicity of the flexible linker sequences is of concern forthe αCD40.LIPO5 HIV peptide vaccine design. The limited datasets shownabove, testing recall of T cells with specificities for epitopes withinthe flexible linker sequences, suggest that the human repertoire againstthese sequences is variable. Also, the ability of these sequences toprime responses de novo is untested. Responses to the αCD40.LIPO5 HIVpeptide vaccine in monkeys can be tested using the present invention. Ifnecessary, certain less desirable epitopes within these regions can beidentified by a combination of predictive computational methods andpeptide stimulation scans, and then eliminated by introducing mutationalchanges that abrogate the TCR interaction.

A humanized antibody includes the heavy chain variable region (V_(H))and a light chain variable region (V_(L)), wherein the framework regionsof the heavy chain and light chain variable regions are from a donorhuman antibody, and wherein the light chain complementarity determiningregions (CDRs) have at least 80%, 90%, 95% or higher identity toCDR1_(L) having the amino acid sequence SASQGISNYLN (SEQ ID NO:41), theCDR2_(L) having the amino acid sequence YTSILHS (SEQ ID NO:42) and theCDR3_(L) having the amino acid sequence QQFNKLPPT (SEQ ID NO:43); andwherein the heavy chain complementarity determining regions comprise atleast 80%, 90%, 95% or higher identity to the CDR1_(H), CDR2_(H) andCDR3_(H), the CDR1_(H) having the amino acid sequence GFTFSDYYMY (SEQ IDNO: 44), the CDR2_(H) having the amino acid sequence YINSGGGSTYYPDTVKG(SEQ ID NO: 45), and the CDR3_(H) having the amino acid sequenceRGLPFHAMDY (SEQ ID NO: 46). For example, the humanized antibody maycomprise a VL framework having at least 95% identity to the framework ofSEQ ID NOS.: 2, 4, 5 or 7 and a VH framework that has at least 95%identity to the framework of SEQ ID NO.:1, 3 or 6. In another aspect,the donor CDR sequences are from anti-CD40_12E12.3F3,anti-CD40_12B4.2C10, anti-CD40_11B6.1C3 or combinations of their heavyor light chains, and/or their variable regions and further, wherein theantibody or fragment thereof specifically binds to CD40.

EXAMPLE 3 Prostate-Specific Antigen (PSA), Cycline D1, MART-1, InfluenzaViral Nucleoprotein (NP) and HA1 Subunit of Influenza ViralHemagglutinin (H1N1, PR8) and Peptide Screen

Internalization of anti-CD40 mAb. 1×10⁶ IL-4DCs were incubated for 1 hin ice with 3 mg/ml human gamma globulin in PBS containing 3% BSA toblock non-specific binding. Cells were pulsed for 30 minutes on ice withAlexa 568 labeled anti-CD40 mAb (all at 20 ng/ml final concentration innon-specific block). Cells were then washed and allowed to internalizesurface bound antibodies for different times, between 0 and 90 minutes,at 37° C. Following internalization, cells were washed twice withice-cold PBS containing 1% BSA and 0.05% sodium azide (PBA) and fixed inice-cold 1% methanol-free formaldehyde (MFF) in PBS overnight at 4° C.Cells were permeablized in PBS 3% BSA containing 0.5% saponin (PBAS) for20 minutes at 4° C., and transferred to a 96-well round bottompolypropylene microtiter plate. After washing twice with ice-cold PBAS,cells were incubated for 1 h on ice with 3 mg/ml human gamma globulin inPBAS. BODIPY-phalloidin diluted in PBAS and incubated with cells for 1hour in ice. Cells were further stained with TOPRO-II, as a nuclearcounterstain. Slides were imaged on a Leica SP1 confocal microscope.

Cells. Monoclonal antibodies for cell surface staining were purchasedfrom BD Biosciences (CA). Monocytes (1×10⁶/ml) from healthy donors werecultured in Cellgenics media (France) containing GM-CSF (100 ng/ml) andIL-4 (50 ng/ml) or GM-CSF (100 ng/ml) and IFNa (500 Units/ml) (R&D, CA).For IFNDCs, cells were fed on day 1 with IFNa and GM-CSF. For IL-4DCs,the same amounts of cytokines were supplemented into the media on dayone and day three. PBMCs were isolated from Buffy coats using Percoll™gradients (GE Healthcare, Buckinghamshire, UK) by density gradientcentrifugation. Total CD4+ and CD8+ T cells were purified by usingStemCell kits (CA).

Peptides. 15-mers (11 amino acid overlapping) for prostate-specificantigen (PSA), Cycline D1, MART-1, influenza viral nucleoprotein (NP)and HA1 subunit of influenza viral hemagglutinin (H1N1, PR8), weresynthesized (Mimotopes).

DCs and T cell co-culture and cytokine expressions. 5×10³ DCs loadedwith recombinant fusion proteins (anti-CD40-HA1, Control Ig-HA1,anti-CD40-PSA, anti-CD40-Cyclin D1, anti-CD40-MART-1, anti-MARCO-MART-1,and control Ig-MART-1) were co-cultured with 2×10⁵ CFSE-labeled CD4+ Tcells for 8 days. Proliferation was tested by measuring CFSE dilutionafter staining cells with anti-CD4 antibody labeled with APC.

For measuring the expression of intracellular IFNγ, CD4+ T cells wererestimulated with 1-5 uM of indicated peptides for 5 h in the presenceof Brefeldin A. In separate experiments, CD4+ T cells were restimulatedwith peptides indicated for 36 h, and then cytokines secreted by CD4+ Tcells were measured by the Luminex.

CD8+ T cells were co-cultured with DCs for 10 days in the presence of 20units/ml IL-2 and 20 units/ml IL-7. On day 10 of the culture, CD8+ Tcells were stained with anti-CD8 and tetramers indicated.

CTL assay. On day 10 of the culture, a 5-h ⁵¹Cr release assay wasperformed. T2 cells pulsed with ⁵¹Cr first and then labeled with 10 uMHLA-A2 epitope of MART-1 or 1 nM epitope of influenza viral M1. T2 cellswithout peptide were used as control. The mean of triplicate samples wascalculated, and the percentage of specific lysis was determined usingthe following formula: percentage of specific lysis=100×(experimental⁵¹Cr release−control ⁵¹Cr release)/(maximum ⁵¹Cr release−control ⁵¹Crrelease). The maximum release refers to counts from targets in 2.5%Triton X-100.

Preparation of mAbs specific for human CD40. Receptor ectodomain.hIgG(human IgG1Fc) and AP (human placental alkaline phosphatase) fusionproteins were produced for immunizing mice and screening mAbs,respectively. A mammalian vector for human IgFc fusion proteins wasengineered as described [J. Immunol. 163: 1973-1983 (1999)]. Themammalian expression vector for receptor ectodomain.AP proteins wasgenerated using PCR to amplify cDNA for AP resides 133-1581(gb|BC009647|) while adding a proximal in-frame Xho I site and a distal6C-terminal His residues followed by a TGA stop codon and Not I site.This Xho I-Not I fragment replaced the human IgG Fc coding sequence inthe above ectodomain.IgG vector. Fusion proteins were produced using theFreeStyle™ 293 Expression System (Invitrogen, CA) according to themanufacturer's protocol (1 mg total plasmid DNA with 1.3 ml 293Fectinreagent/L of transfection). Receptor ectodomain.hIgG was purified by 1ml HiTrap protein A affinity chromatography (GE Healthcare, CA) elutedwith 0.1 M glycine, pH 2.7. Fractions were neutralized with 2M Tris, andthen dialyzed against PBS.

Mouse mAbs were generated by conventional technology. Briefly,six-week-old BALB/c mice were immunized i.p. with 20 μg of receptorectodomain.hIgGFc fusion protein with Ribi adjuvant, then boosted with20 μg antigen ten days and fifteen days later. After three months, themice were boosted again three days prior to taking the spleens. Three tofour days after a final boosting, draining lymph nodes (LN) wereharvested. B cells from spleen or LN cells were fused with SP2/O—Ag 14cells (ATCC). Hybridoma supernatants were screened to analyze mAbsspecific to the receptor ectodomain fusion protein compared to thefusion partner alone, or to the receptor ectodomain fused to alkalinephosphatase [J. Immunol. 163: 1973-1983 (1999)]. Positive wells werethen screened in FACS using 293F cells transiently transfected withexpression plasmids encoding full-length receptor cDNAs. Selectedhybridomas were single cell cloned and expanded in CELLine flasks(Integra, CA). Hybridoma supernatants were mixed with an equal volume of1.5 M glycine, 3 M NaCl, 1×PBS, pH 7.8 (binding buffer) and tumbled withMabSelect resin (GE Healthcare, CA) (800 ml/5 ml supernatant). The resinwas washed with binding buffer and eluted with 0.1 M glycine, pH 2.7.Following neutralization with 2 M Tris, mAbs were dialyzed against PBS.

Expression and purification of recombinant mAbs. Total RNA was preparedfrom hybridoma cells using RNeasy kit (Qiagen, CA) and used for cDNAsynthesis and PCR (SMART RACE kit, BD Biosciences) using supplied 5′primers and gene specific 3′ primers (mIgGκ,5′ggatggtgggaagatggatacagttggtgcagcatc3′ (SEQ ID NO.:48); mIgG2a,5′ccaggcatcctagagtcaccgaggagccagt3′) (SEQ ID NO.:49). PCR products werethen cloned (pCR2.1 TA kit, Invitrogen) and characterized by DNAsequencing (MC Lab, CA). Using the derived sequences for the mouse heavy(H) and light (L) chain variable (V)-region cDNAs, specific primers wereused to PCR amplify the signal peptide and V-regions while incorporatingflanking restriction sites for cloning into expression vectors encodingdownstream human IgGκ or IgG4H regions. The vector for expression ofchimeric mVκ-hIgκ was built by amplifying residues 401-731(gi|63101937|) flanked by Xho I and Not I sites and inserting this intothe Xho I-Not I interval of pIRES2-DsRed2 (BD Biosciences). PCR was usedto amplify the mAb Vk region from the initiator codon, appending a Nhe Ior Spe I site then CACC, to the region encoding (e.g., residue 126 ofgi|76779294|), appending a distal Xho I site. The PCR fragment was thencloned into the Nhe I-Not I interval of the above vector. The controlhuman IgGκ sequence corresponds to gi|49257887| residues 26-85 andgi|21669402| residues 67-709. The control human IgG4H vector correspondsto residues 12-1473 of gi|19684072| with S229P and L236E substitutions,which stabilize a disulphide bond and abrogate residual FcR interaction[J. Immunol. 164: 1925-1933 (2000)], inserted between the Bgl II and NotI sites of pIRES2-DsRed2 while adding the sequence 5′gctagctgattaattaa3′ instead of the stop codon. PCR was used to amplify the mAb VH regionfrom the initiator codon, appending CACC then a Bgl II site, to theregion encoding residue 473 of gi|19684072|. The PCR fragment was thencloned into the Bgl II-Apa I interval of the above vector.

Expression and purification of Flu HA1 fusion protein. The Flu HA1antigen coding sequence is a CipA protein [Clostridium. thermocellum]gi|479126| residues 147-160 preceding hemagglutinin [Influenza A virus(A/Puerto Rico/8/34(H1N1))] gi|126599271| residues 18-331 with a P321Lchange and with 6 C-terminal His residues was inserted between the Heavychain vector Nhe I and Not I sites to encode recombinant antibody-HA1fusion proteins (rAb.HA1). Similarly, recombinant antibody-PSA fusionproteins (rAb.PSA) were encoded by inserting gi|4784812| prostatespecific antigen residues 101-832 with proximal sequenceGCTAGCGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACACTT CTAGCGC(SEQ ID NO.:27) (Nhe I site and CipA spacer) and a distal Not I siteinto the same Heavy chain vector. Recombinant antibody proteins wereexpressed and purified as described above for hFc fusion proteins. Insome cases the rAb.antigen coding region and the corresponding L chaincoding region were transferred to separate cetHS-puro UCOE vectors(Millipore, CA). The use of UCOE vectors in combination with apreadapted serum free, suspension cell line allowed for rapid productionof large quantities of protein [Cytotechnology 38, 43-46 (2002).] CHO-Scells grown in CD-CHO with GlutaMAX and HT media supplement (Invitrogen)were seeded at 5×10⁵ ml 24 h prior to transfection in 500 ml CorningEhrlenmyer flasks and incubated in 8% CO₂ at 125 rpm. On the day oftransfection, 1.2×10⁷ cells with viability at least 95% were added to afinal volume of 30 ml in a 125 ml flask in CD-CHO with GlutaMAX. 48 mlof FreeStyle Max reagent (Invitrogen) in 0.6 ml of OptiPRO SFM(Invitrogen) was added with gentle mixing to 24 mg of Sce I-linearizedlight chain vector and 24 mg of Sce I-linearized Heavy chain vectormixed and sterile filtered in 0.6 ml of OptiPRO SFM. After 20 min, theDNA-lipid complex was slowly added to the 125 ml CHO-S culture flaskwith swirling. Cells were incubated 24 h before adding 30 ml of acombined media solution of CD-CHO with CHO-M5 (Sigma, C0363 component ofCHO Kit 1) containing 5 mg/ml of puromycin (A.G. Scientific, CA),2×GlutaMAX and 0.25×Pen/Strep (Invitrogen). At day 2, another 5 mg/ml ofpuromycin was added directly to the culture and selection was allowed toproceed ˜10-14 days while following cell viability from six days posttransfection. The viable cell count dropped and when the viable densityis ˜2-3×10⁶/ml, the cells were transferred to fresh selection medium (CDCHO-S+CHO M5 with 2×GlutaMAX, 0.25×Pen/Strep, 10 mg/ml Puromycin) at1E6/ml. Frozen cell stocks were prepared when viability reached >90%.Cells were split in selection medium when cell density exceeded 2×10⁶/mluntil scaled to 4×250 ml in 500 ml flasks. Supernatant was harvestedwhen cell viability dropped below 80% with a maximum final cell density7×10⁶/ml. Endotoxin levels were less than 0.2 units/ml.

Expression and purification of recombinant Flu M1 and MART-1 proteins.PCR was used to amplify the ORF of Influenza A/Puerto Rico/8/34/MountSinai (H1N1) M1 gene while incorporating an Nhe I site distal to theinitiator codon and a Not I site distal to the stop codon. The digestedfragment was cloned into pET-28b(+) (Novagen), placing the M1 ORFin-frame with a His6 tag, thus encoding His.Flu M1 protein. A pET28b (+)derivative encoding an N-terminal 169 residue cohesin domain from C.thermocellum (unpublished) inserted between the Nco I and Nhe I sitesexpressed Coh.His. For expression of Cohesin-Flex-hMART-1-PeptideA-His,the sequenceGACACCACCGAGGCCCGCCACCCCCACCCCCCCGTGACCACCCCCACCACCACCGACCGGAAGGGCACCACCGCCGAGGAGCTGGCCGGCATCGGCATCCTGACCGTGATCCTGGGCGGCAAGCGGACCAACAACAGCACCCCCACCAAGGGCGAATTCTGCAGATATCCATCACACTGGCGGCCG (SEQ IDNO.:28) (encodingDTTEARHPHPPVTTPTTDRKGTTAEELAGIGILTVILGGKRTNNSTPTKGEFCRYPSHWRP (SEQ IDNO.:29)—the italicized residues are the immunodominant HLA-A2-restrictedpeptide and the underlined residues surrounding the peptide are fromMART-1) was inserted between the Nhe I and Xho I sites of the abovevector. The proteins were expressed in E. coli strain BL21 (DE3)(Novagen) or T7 Express (NEB), grown in LB at 37° C. with selection forkanamycin resistance (40 μg/ml) and shaking at 200 rounds/min to mid logphase growth when 120 mg/L IPTG was added. After three hours, the cellswere harvested by centrifugation and stored at −80° C. E. coli cellsfrom each 1 L fermentation were resuspended in 30 ml ice-cold 50 mMTris, 1 mM EDTA pH 8.0 (buffer B) with 0.1 ml of protease inhibitorCocktail II (Calbiochem, CA). The cells were sonicated on ice 2×5 min atsetting 18 (Fisher Sonic Dismembrator 60) with a 5 min rest period andthen spun at 17,000 r.p.m. (Sorvall SA-600) for 20 min at 4° C. ForHis.Flu M1 purification the 50 ml cell lysate supernatant fraction waspassed through 5 ml Q Sepharose beads and 6.25 ml 160 mM Tris, 40 mMimidazole, 4 M NaCl pH 7.9 was added to the Q Sepharose flow through.This was loaded at 4 ml/min onto a 5 ml HiTrap chelating HP columncharged with Ni++. The column-bound protein was washed with 20 mM NaPO₄,300 mM NaCl pH 7.6 (buffer D) followed by another wash with 100 mMH₃COONa pH 4.0. Bound protein was eluted with 100 mM H₃COONa pH 4.0. Thepeak fractions were pooled and loaded at 4 ml/min onto a 5 ml HiTrap Scolumn equilibrated with 100 mM H₃COONa pH 5.5, and washed with theequilibration buffer followed by elution with a gradient from 0-1 M NaClin 50 mM NaPO₄ pH 5.5. Peak fractions eluting at about 500 mM NaCl werepooled. For Coh.Flu M1.His purification, cells from 2 L of culture werelysed as above. After centrifugation, 2.5 ml of Triton X114 was added tothe supernatant with incubation on ice for 5 min. After furtherincubation at 25° C. for 5 min, the supernatant was separated from theTriton X114 following centrifugation at 25° C. The extraction wasrepeated and the supernatant was passed through 5 ml of Q Sepharosebeads and 6.25 ml 160 mM Tris, 40 mM imidazole, 4 M NaCl pH 7.9 wasadded to the Q Sepharose flow through. The protein was then purified byNi⁺⁺chelating chromatography as described above and eluted with 0-500 mMimidazole in buffer D.

FIG. 13 shows the internalization of anti-CD40 mAb:IL-4DC. IL-4DCs weretreated with 500 ng/ml of anti-CD40-Alexa 568. FIG. 14 shows CD4 and CD8T cell proliferation by DCs targeted with anti-CD40-HAL 5×10e3 IFNDCsloaded with 2 ug/ml of anti-CD40-HA or control Ig-HA1 were co-culturedwith CFSE-labeled autologous CD4+ or CD8+ T cells (2×10e5) for 7 days.Cells were then stained with anti-CD4 or anti-CD8 antibodies. Cellproliferation was tested by measuring CFSE-dilution. FIG. 15 shows atitration of HA1 fusion protein on CD4+ T proliferation. IFNDCs (5K)loaded with fusion proteins were co-cultured with CFSE-labeled CD4+ Tcells (200K) for 7 days. FIG. 16 shows IFNDCs targeted withanti-CD40-HA1 activate HA1-specific CD4+ T cells. CD4+ T cells wererestimulated with DCs loaded with 5 uM of indicated peptides, and thenintracellular IFN-γ was stained. FIG. 17 shows IFNDCs targeted withanti-CD40-HA1 activate HA1-specific CD4+ T cells. CD4+ T cells wererestimulated with DCs loaded with indicated peptides for 36 h, and thenculture supernatant was analyzed for measuring IFN-γ. FIG. 18 shows thattargeting CD40 results in enhanced cross-priming of MART-1 specific CD8+T cells. IFNDCs (5K/well) loaded with fusion proteins were co-culturedwith purified CD8+ T cells for 10 days. Cells were stained with anti-CD8and tetramer. Cells are from healthy donors (HLA-A*0201+). FIG. 19 showstargeting CD40 results in enhanced cross-priming of MART-1 specific CD8+T cells (Summary of 8-repeated experiments using cells from differenthealthy donors). FIG. 20 shows CD8+ CTL induced with IFNDCs targetedwith anti-CD40-MART-1 are functional. CD8+ T cells co-cultured withIFNDCs targeted with fusion proteins were mixed with T2 cells loadedwith 10 uM peptide epitope. FIG. 21 shows CD8+ CTL induced with IFNDCstargeted with anti-CD40-Flu M1 are functional. CD8+ T cells co-culturedwith IFNDCs targeted with fusion proteins were mixed with T2 cellsloaded with 1.0 nM peptide epitope. FIG. 22 shows an outline of protocolto test the ability a vaccine composed of anti-CD4012E12 linked to PSA(prostate specific antigen) to elicit the expansion from a naïve T cellpopulation. PSA-specific CD4+ T cells corresponding to a broad array ofPSA epitopes. Briefly, DCs derived by culture with IFNα and GM-CSF ofmonocytes from a healthy donor are incubated with the vaccine. The nextday, cells are placed in fresh medium and pure CD4+ T cells from thesame donor are added. Several days later, PSA peptides are added and,after four hours, secreted gamma-IFN levels in the culture supernatantsare determined.

FIG. 23 shows that many PSA peptides elicit potent gamma-IFN-productionresponses indicating that anti-CD4012E12 and similar anti-CD40 agentscan efficiently deliver antigen to DCs, resulting in the priming ofimmune responses against multiple epitopes of the antigen. The peptidemapping of PSA antigens. 5×10e3 IFNDCs loaded with 2 ug/ml ofanti-CD40-PSA were co-cultured with purified autologous CD4+ T cells(2×10e5) for 8 days. Cells were then restimulated with 5 uM ofindividual peptides derived from PSA for 36 h. The amount of IFNγ wasmeasured by Luminex Cells are from healthy donors.

FIG. 24 shows DCs targeted with anti-CD40-PSA induce PSA-specific CD8+ Tcell responses. IFNDCs were targeted with 1 ug mAb fusion protein withPSA. Purified autologous CD8+ T cells were co-cultured for 10 days.Cells were stained with anti-CD8 and PSA (KLQCVDLHV—SEQ ID NO:131)-tetramer. Cells are from a HLA-A*0201 positive healthy donor. Theresults demonstrate that anti-CD40 effectively deliver PSA to the DCs,which in turn elicit the expansion of PSA-specific CD8+ T cells.Briefly, 5×10e3 IFNDCs loaded with 2 ug/ml of anti-CD40-PSA wereco-cultured with purified autologous CD8+ T cells (2×10e5) for 10 days.Cells were then stained with tetramer. Cells are from HLA-0*201 positivehealthy donor.

FIG. 25 a scheme (left) and the IFN-γ production by T cells of the poolsof peptides and control for Donor 2. 5×10e3 IFNDCs loaded with 2 ug/mlof anti-CD40-Cyclin D1 were co-cultured with purified autologous CD4+ Tcells (2×10e5) for 8 days. Cells were then restimulated with 5 uM ofindividual peptides derived from CyclinD1 for 5 h in the presence ofBrefeldin A. Cells were stained for measuring intracellular IFNγexpression.

FIG. 26 shows a peptide scan and IFN-γ production by T cells obtainedfrom the pools of peptides shown in FIG. 25 and control for Donor 2.5×10e3 IFNDCs loaded with 2 ug/ml of anti-CD40-Cyclin D1 wereco-cultured with purified autologous CD4+ T cells (2×10e5) for 8 days.Cells were then restimulated with 5 uM of individual peptides derivedfrom CyclinD1 for 5 h in the presence of Brefeldin A. Cells were stainedfor measuring intracellular IFNγ expression.

In conclusion, delivering antigens to DCs, the most potent antigenpresenting cells, via CD40 is an efficient way to induce and activateantigen specific both CD4+ and CD8+ T cell-mediated immunity. Thus,vaccines made of anti-CD40 mAb will induce potent immunity againstcancer and infections.

Peptide information: HA1 sequences: (SEQ ID NO.: 30)MKANLLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL EDSHNGKLCR(SEQ ID NO.: 31) LKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELRE (SEQ ID NO.: 32)QLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEK EGSYPKLKNS(SEQ ID NO.: 33) YVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQA (SEQ ID NO.: 34)GRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHE CNTKCQTPLG(SEQ ID NO.: 35) AINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSISequences of peptides in FIG. 17 (SEQ ID NO.: 36)Peptide 22: SSFERFEIFPKESSWPN (SEQ ID NO.: 37)Peptide 45: GNLIAPWYAFALSRGFG (SEQ ID NO.: 38)Peptide 46: WYAFALSRGFGSGIITS NP sequences: (SEQ ID NO.: 39)MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLS DYEGRLIQNS(SEQ ID NO.: 13) LTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIW (SEQ ID NO.: 125)RQANNGDDATAGLTHMMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGS TLPRRSGAAG(SEQ ID NO.: 126) AAVKGVGTMVMELVRMIKRGINDRNFWRGENGRKTRIAYERMCNILKGKFQTAAQKAMMD (SEQ ID NO.: 127)QVRESRNPGNAEFEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYD FEREGYSLVG(SEQ ID NO.: 128) IDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGTKVLPRGKLST (SEQ ID NO.: 129)RGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISIQP TFSVQRNLPF(SEQ ID NO.: 130) DRTTIMAAFNGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKAASPIVPSFD (SEQ ID NO.: 40) MSNEGSYFFGDNAEEYDNSequences of peptides in FIG. 23 (SEQ ID NO.: 47)Peptide 22: GKWVRELVLYDKEEIRR (SEQ ID NO.: 50)Peptide 33: RTGMDPRMCSLMQGSTL (SEQ ID NO.: 51)Peptide 46: MCNILKGKFQTAAQKAM Prostate specific antigen (PSA) sequence(SEQ ID NO.: 52) MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWV (SEQ ID NO.: 53)LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRF LRPGDDSSHD(SEQ ID NO.: 54) LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVIS (SEQ ID NO.: 55)NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWG SEPCALPERP(SEQ ID NO.: 56) SLYTKVVHYRKWIKDTIVANP Sequences of peptides in FIG. 23(SEQ ID NO.: 57) Peptide 1: APLILSRIVGGWECE (SEQ ID NO.: 58)Peptide 4: ECEKHSQPWQVLVAS (SEQ ID NO.: 59) Peptide 25: GDDSSHDLMLLRLSE(SEQ ID NO.: 60) Peptide 26: SHDLMLLRLSEPAEL (SEQ ID NO.: 61)Peptide 49: SGDSGGPLVCNGVLQ (SEQ ID NO.: 62) Peptide 54: GSEPCALPERPSLYT(SEQ ID NO.: 63) Peptide 56: ERPSLYTKVVHYRKW (SEQ ID NO.: 64)Peptide 58: VVHYRKWIKDTIVAN Cyclin D1 sequence (SEQ ID NO.: 65)MRSYRFSDYLHMSVSFSNDMDLFCGEDSGVFSGESTVDFSSSEVDSWPGD SIACFIEDER(SEQ ID NO.: 66) HFVPGHDYLSRFQTRSLDASAREDSVAWILKVQAYYNFQPLTAYLAVNYMDRFLYARRLP (SEQ ID NO.: 67)ETSGWPMQLLAVACLSLAAKMEEILVPSLFDFQVAGVKYLFEAKTIKRME LLVLSVLDWR(SEQ ID NO.: 68) LRSVTPFDFISFFAYKIDPSGTFLGFFISHATEIILSNIKEASFLEYWPSSIAAAAILCV (SEQ ID NO.: 69)ANELPSLSSVVNPHESPETWCDGLSKEKIVRCYRLMKAMAIENNRLNTPK VIAKLRVSVR(SEQ ID NO.: 70) ASSTLTRPSDESSFSSSSPCKRRKLSGYSWVGDETSTSNSequences of peptides in FIG. 26. (SEQ ID NO.: 71)Peptide 7: DRVLRAMLKAEETCA (SEQ ID NO.: 72) Peptide 8: RAMLKAEETCAPSVS(SEQ ID NO.: 73) Peptide 10: TCAPSVSYFKCVQKE

MART-1 Antigen. MART-1 is a tumor-associated melanocytic differentiationantigen. Vaccination with MART-1 antigen may stimulate a host cytotoxicT-cell response against tumor cells expressing the melanocyticdifferentiation antigen, resulting in tumor cell lysis.

FIG. 27 shows the expression and construct design for anti-CD40-MART-1peptide antibodies. FIG. 28 is a summary of the CD4⁺ and CD8⁺immunodominant epitopes for MART-1. FIGS. 27 and 28 show the use of theflexible linker technology to permit the successful expression ofrecombinant anti-DC receptor targeting antibodies fused to significant(˜⅔) parts of human MART-1. Recombinant antibody fused at the Heavychain C-terminus to the entire MART-1 coding region is not at allsecreted from production mammalian cells [not shown]. TheFlex-v1-hMART-1-Pep-3-f4-Pep-1 adduct is particularly well expressed andis one preferred embodiment of a MART-1-targeting vaccine, as is theFlex-v1-hMART-1-Pep-3-4-Pep-1-f3-Pep-2 adduct which bears a maximum loadof MART-1 epitopes. Slide 2 of the MART-1 powerpoint presentation showsthat these adducts can be successfully appended to multiple anti-DCreceptor vehicles.

The sequence below is a Heavy chain—hMART-1 peptides string ofpep3-pep1-pep2 fusion protein where each hMART1 peptide sequence[bold-italics] is separated by a inter-peptide spacer f [shown in bold].In this case, a 27-amino-acid long linker flex-v1(v1) [italics] derivedfrom cellulosomal anchoring scaffoldin B precursor [Bacteroidescellulosolvens—described in the gag-nef vaccine invention disclosure]was inserted between the Heavy chain C-terminus and the hMART1peptides-flexible spaces string. The underlined AS residues are joiningsequences.

[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-1]C981 is: (SEQ ID NO.: 74)EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP AS

AS TNGSITVAATAPTVTPTVNATPSAA AS

A

[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-344-Pep-143 -Pep-2]C978 is: (SEQ ID NO.: 75)EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP AS

AS TNGSITVAATAPTVTPTVNATPSAA AS

AS TVTPTATATPSAI VTTITPTATTKP AS

AS [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-1]C1012 is: (SEQ ID NO.: 76)QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP AS

AS TNGSITVAATAPTVTPTVNATPSA A AS

AS [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-143-Pep-2]C1013 is: (SEQ ID NO.: 77)QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP AS

AS TNGSITVAATAPTVTPTVNATPSAA AS

AS TVTPTATATP SAIVTTITPTATTKP AS

AS MART-1 DNA Sequence: MART-1 constructs with 3 peptides, Start/stopsites are underlined, peptide 1 is bold, peptide 2is bold-italics and peptide 3 is bold-underlined: (SEQ ID NO.: 78)AACACCGACAACAACAGATGATCTGGATGCAGCTAGT GGGTTTGATCATCGGGACAGCAAAGTGTCTCTTCAAGAGAAAAACTGTGAACCTGTGGTTCCCAATGCTCCACCTGCTTATGAGAAACTCTCTGCAGAACAGTCACCACCACCTTATTCACCTGCTAGTACCAACGGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGT

A

GCTGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGACCACCATCACCCCCACCGCCACCACCAAGCCCGCTAGT GTCTTACTGCTCATCGGCTGTTGGTATTGTAGAAGACGAAATGGATACAGAGCCTTGATGGATAAAAGTCTTCATGTTGGCACTCAATGTGCCTTAACAAGAAGATGCCCACAAGAAGGGtgaGCGGCCGCATCGAAGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCMART1-Peptide 3, the italicized portion is the CD4+immunodominant epitope. (SEQ ID NO.: 79)GFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSP Flex-4 (SEQ ID NO.: 80)ASTNGSITVAATAPTVTPTVNATPSAAASMART1-Peptide 1 the italicized portion is the CD4+immunodominant epitope and the underlined-italicized portion is the CD8+ immunodominant epitope (SEQ ID NO.: 81)MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILG Flex-3: (SEQ ID NO.: 82)ASTVTPTATATPSAIVTTITPTATTKPASMART1-Peptide 2 the italicized portion is the CD4+immunodominant epitope. (SEQ ID NO.: 83)VLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGMART1 constructs with two peptides:Peptide 3 is bold-italics-underlined, flex-4 isbold and Peptide 1 is bold-italics-underlined: (SEQ ID NO.: 84)

ASTNGSITV AATAPTVTPTVNATPSAAAS

AS Protein Sequence: C978. rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3(bold-italics-underlined)-f4 (bold)-Pep-1 (bold-italics)-f3 (italics)-Pep-2 (bold-underlined)] (SEQ ID NO.: 85)MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPAS

ASTNGSITVAATA ASTNGSITVAATAPTVTPTVNATPSAAAS

ASTVTPTATATPSAIVTTITPTATTKPAS VLLLIGCWYCRRRNGYRALMDKSLHVGTCQCALTRRCPQEGASProtein Sequence: C981. rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3(bold-italics-underlined)-f4-(bold)-Pep-1](bold- underlined)(SEQ ID NO.: 86) MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPAS

ASTNGSITVAATA PTVTPTVNATPSAAAS MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILT VILGAS

GP100 Antigen. GP100 antigen is a melanoma-associated antigen. Whenadministered in a vaccine formulation, gp100 antigen may stimulate acytotoxic T cell HLA-A2.1-restricted immune response against tumors thatexpress this antigen, which may result in a reduction in tumor size.

GP100 ectodomain coding region fused to recombinant antibody Heavy chaincoding region is not at all secreted by production mammalian cells. Thetotal sequence is shown below—italics residues are the leader sequenceand the transmembrane domain, the peptides are in bold-italics and thetransmembrane domain is italics-underlined.

(SEQ ID NO.: 87) MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFV YVW

LGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPL AHSSSAFT

SVSQLRALDGGNKHFLRNQPLTFALQLHDPSGY LAEADLSYTWDFGDSSGTLISRALVVTHTY

QVVLQAAIPLT SCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQ VPLI VGILLVLMAVVLASIYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIG ENSPLLSGQQV

Known HLA-A0201 restricted peptides sequences are: GP100 M: 209-217(2M): IMDQVPFSV (SEQ ID NO.:88); 209-217 WT: ITDQVPFSV (SEQ ID NO.:89)GP100 M: 280-288 (9V): YLEPGPVTV (SEQ ID NO.:90) 280-288 WT: YLEPGPVTA(SEQ ID NO.:91) GP100 WT: 154-162: KTWGQYWQV (SEQ ID NO.:92)

FIG. 29-33 show the gp100 adducts which were successfully expressed assecreted anti-DC receptor targeting vaccines. These employed the use ofthe flexible linker sequences and fragmentation and shuffling of thegp100 ectodomain coding region. Preferred embodiments of gp100 vaccineadducts are described.

FIG. 29 shows the expression and construct design for anti-CD40-gp100peptide antibodies. FIG. 30 shows the design for additionalanti-CD40-gp100 peptide antibodies. FIG. 31 shows the expression andconstruct design for additional anti-CD40-gp100 peptide antibodies. FIG.32 is a summary of the CD4⁺ and CD8⁺ immunodominant epitopes for gp100.FIG. 33 shows the expression and construct design for additionalanti-CD40-gp100 peptide antibodies.

rAB-cetHS-puro[ manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-hgp100-Pep-3-f3-Pep-4-f4-Pep-5-f3-Pep-2 ] C1285,the peptides are bold-italics, flexible linkers arebold and the underlined AS residues are joining sequences:(SEQ ID NO.: 93) EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS

AS TNGSITVAATAPTVTPTVN ATPSAA AS

AS TVTPTATATPSAIVTTITPTATTKPAS

AS TNGSITVAATAPTVTPTVNATPSAA AS

AS TVTPTATATPSAIVTTITPTATTKP AS

AS rAB-cetHS-puro[hIgG4H-C-Flex-hgp100-Pep-1-f4-Pep-3-f3-Pep-4-f4-Pep-5-f3-Pep-2] C1286: (SEQ ID NO.: 94)RLQLQESGPGLLKPSVTLSLTCTVSGDSVASSSYYWGWVRQPPGKGLEWIGTINFSGNMYYSPSLRSRVTMSADMSENSFYLKLDSVTAADTAVYYCAAGHLVMGFGAHWGQGKLVSVSPASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS

AS TNGSITVAATAPTVTPTVN ATPSAA AS

AS TVTPTATATPSAIVTTITPTATTKP AS

AS TNGSITVAATAPTVTPTVNATPSAA AS

AS TVTPTATATPSAIVTTITPTATTKP AS

AS

gp100: —Nucleic Acid Sequence. Peptide 1—underlined, Peptide 2—italics,Peptide 3—bold, Peptide 4—bold-underlined, Peptide 5 bold-italics.

(SEQ ID NO.: 95) GATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAAAAGTACCCAGAAACCAGGACTGGCTTGGTGTCTCAAGGCAACTCAGAACCAAAGCCTGGAACAGGCAGCTGTATCCAGAGTGGACAGAAGCCCAGAGACTTGACTGCTGGAGAGGTGGTCAAGTGTCCCTCAAGGTCAGTAATGATGGGCCTACACTGATTGGTGCAAATGCCTCCTTCTCTATTGCCTTGAACTTCCCTGGAAGCCAAAAGGTATTGCCAGATGGGCAGGTTATCTGGGTCAACAATACCATCATCAATGGGAGCCAGGTGTGGGGAGGACAGCCAGTGTATCCCCAGGAAACTGACGATGCCTGCATCTTCCCTGATGGTGGACCTTGCCCATCTGGCTCTTGGTCTCAGAAGAGAAGCTTTGTTTATGTCTGGAAGACCTGGGGCCAATACTGGCAAGTTCTAGGGGGCCCAGTGTCTGGGCTGAGCATTGGGACAGGCAGGGCAATGCTGGGCACACACACCATGGAAGTGACTGTCTACCATCGCCGGGGATCCCAGAGCTATGTGCCTCTTGCTCATTCCAGCTCAGCCTTCACCATTACTGACCAGGTGCCTTTCTCCGTGAGCGTGTCCCAGTTGCGGGCCTTGGATGGAGGGAACAAGCACTTCCTGAGAAATCAGGCTAGTACCAACGGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGTGGCACCACAGATGGGCACAGGCCAACTGCAGAGGCCCCTAACACCACAGCTGGCCAAGTGCCTACTACAGAAGTTGTGGGTACTACACCTGGTCAGGCGCCAACTGCAGAGCCCTCTGGAACCACATCTGTGCAGGTGCCAACCACTGAAGTCATAAGCACTGCACCTGTGCAGATGCCAACTGCAGAGAGCACAGGTATGACACCTGAGAAGGTGCCAGTTTCAGAGGTCATGGGTACCACACTGGCAGAGATGTCAACTCCAGAGGCTACAGGTATGACACCTGCAGAGGTATCAATTGTGGTGCTTTCTGGAACCACAGCTGCAGCTAGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGACCACCATCACCCCCACCGCCACCACCAAGCCCGCTAGTCAGGTAACAACTACAGAGTGGGTGGAGACCACAGCTAGAGAGCTACCTATCCCTGAGCCTGAAGGTCCAGATGCCAGCTCAATCATGTCTACGGAAAGTATTACAGGTTCCCTGGGCCCCCTGCTGGATGGTACAGCCACCTTAAGGCTGGTGAAGAGACAAGTCCCCCTGGATTGTGTTCTGTATCGATATGGTTCCTTTTCCGTCACCCTGGACATTGTCCAGGCTAGTACCAACGGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCT AGTGGTATTGAAAGTGCCGAGATCCTGCAGGCTGTGCCGTCCGGTGAGGGGGATGCATTTGAGCTGACTGTGTCCTGCCAAGGCGGGCTGCCCAAGGAAGCCTGCATGGAGATCTCATCGCCAGGGTGCCAGCCCCCTGCCCAGCGGCTGTGCCAGCCTGTGCTACCCAGCCCAGCCTGCCAGCTGGTTCTGCACCAGATACTGAAGGGTGGCTCGGGGACATACTGCCTCAATGTGTCTCTGGCTGATACCAACAGCCTGGCAGTGGTCAGCACCCAGCTTATCGTGCCTGGGATTCTTCTCACAGGTCAAGAAGCAGGCCTTGGGCAGTAA GCTAGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGACCACCATCACCCCCACCG CCACCACCAAGCCCGCTAGT

 GCTAGC  TGA GP100-Peptide 1-Nucleic Acid Sequence. (SEQ ID NO.: 96)GATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAAAAGTACCCAGAAACCAGGACTGGCTTGGTGTCTCAAGGCAACTCAGAACCAAAGCCTGGAACAGGCAGCTGTATCCAGAGTGGACAGAAGCCCAGAGACTTGACTGCTGGAGAGGTGGTCAAGTGTCCCTCAAGGTCAGTAATGATGGGCCTACACTGATTGGTGCAAATGCCTCCTTCTCTATTGCCTTGAACTTCCCTGGAAGCCAAAAGGTATTGCCAGATGGGCAGGTTATCTGGGTCAACAATACCATCATCAATGGGAGCCAGGTGTGGGGAGGACAGCCAGTGTATCCCCAGGAAACTGACGATGCCTGCATCTTCCCTGATGGTGGACCTTGCCCATCTGGCTCTTGGTCTCAGAAGAGAAGCTTTGTTTATGTCTGGAAGACCTGGGGCCAATACTGGCAAGTTCTAGGGGGCCCAGTGTCTGGGCTGAGCATTGGGACAGGCAGGGCAATGCTGGGCACACACACCATGGAAGTGACTGTCTACCATCGCCGGGGATCCCAGAGCTATGTGCCTCTTGCTCATTCCAGCTCAGCCTTCACCATTACTGACCAGGTGCCTTTCTCCGTGAGCGTGTCCCAGTTGCGGGCCTTGGATGGAGGGAACAAGCACTTCCTGAGAAATCAG Protein Sequence:(SEQ ID NO.: 97) DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQ GP100-Peptide 3 (SEQ ID NO.: 98)GGCACCACAGATGGGCACAGGCCAACTGCAGAGGCCCCTAACACCACAGCTGGCCAAGTGCCTACTACAGAAGTTGTGGGTACTACACCTGGTCAGGCGCCAACTGCAGAGCCCTCTGGAACCACATCTGTGCAGGTGCCAACCACTGAAGTCATAAGCACTGCACCTGTGCAGATGCCAACTGCAGAGAGCACAGGTATGACACCTGAGAAGGTGCCAGTTTCAGAGGTCATGGGTACCACACTGGCAGAGATGTCAACTCCAGAGGCTACAGGTATGACACCTGCAGAGGTATCAATTGTGGTGCTTTCTGGAACCACAGCTGCA Protein Sequence: (SEQ ID NO.: 99)GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSI VVLSGTTAAGP100-Peptide 4: (SEQ ID NO.: 100)CAGGTAACAACTACAGAGTGGGTGGAGACCACAGCTAGAGAGCTACCTATCCCTGAGCCTGAAGGTCCAGATGCCAGCTCAATCATGTCTACGGAAAGTATTACAGGTTCCCTGGGCCCCCTGCTGGATGGTACAGCCACCTTAAGGCTGGTGAAGAGACAAGTCCCCCTGGATTGTGTTCTGTATCGATATGGTTCCTTTTCCGTCACCCTGGACATTGTCCAG Protein Sequence: (SEQ ID NO.: 101)QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQ GP100-Peptide 5 (SEQ ID NO.: 102)GGTATTGAAAGTGCCGAGATCCTGCAGGCTGTGCCGTCCGGTGAGGGGGATGCATTTGAGCTGACTGTGTCCTGCCAAGGCGGGCTGCCCAAGGAAGCCTGCATGGAGATCTCATCGCCAGGGTGCCAGCCCCCTGCCCAGCGGCTGTGCCAGCCTGTGCTACCCAGCCCAGCCTGCCAGCTGGTTCTGCACCAGATACTGAAGGGTGGCTCGGGGACATACTGCCTCAATGTGTCTCTGGCTGATACCAACAGCCTGGCAGTGGTCAGCACCCAGCTTATCGTGCCTGGGATTCTTCTCACAGGTCAAGAAGCAGGCCTTGGGCAG Protein Sequence: (SEQ ID NO.: 103)GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIVPGILL TGQEAGLGQGP100-Peptide 2 (SEQ ID NO.: 104)CCTCTGACCTTTGCCCTCCAGCTCCATGACCCTAGTGGCTATCTGGCTGAAGCTGACCTCTCCTACACCTGGGACTTTGGAGACAGTAGTGGAACCCTGATCTCTCGGGCACYTGTGGTCACTCATACTTACCTGGAGCCTGGCCCAGTCACTGCCCAGGTGGTCCTGCAGGCTGCCATTCCTCTCACCTCCTGTGGCTC CTCCCCAGTTCCAGCTAGCProtein Sequence: (SEQ ID NO.: 105)PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPAS

Cyclin B1 Antigen. Cyclin B1, also known as CCNB1, is a human gene thatencodes a regulatory protein involved in mitosis. Cyclin B1 complexeswith p34(cdc2) to form the maturation-promoting factor (MPF). Twoalternative transcripts are known that are the result of alternativetranscription initiation sites. A first transcript encodes aconstitutively expressed transcript. The second transcript is a cellcycle-regulated transcript expressed predominantly during G2/M phase.

The following amino acid sequence is human cyclin B1. Two peptideregions known to contain T cell epitopes are highlighted inbold-underlined and italics-underlined.

(SEQ ID NO.: 106) MALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTALGDIGNKVSEQLQAKMPMKKEAKPSATGKVIDKKLPKPLEKVPMLVPVPVSEPVPEPEPEPEPEPVKEEKLSPEPILVDTASPSPMETSGCAPAEEDLCQAFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEEQAVRPKYLLGREVTGN MRAILIDWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK MLQLVGVTAMFIASKYEEMYPPEIGDFAFVTDNTYTKHQIRQ

DMVHFPPSQIAAGAFC LALKILDNGEWTPTLQHYLSYTEESLLPVMQHLAKNVVMVNQGLTKHMTVKNKYATSKHAKISTLPQLNSALVQDLAKAVAKVHHHHHH Peptide-1 (SEQ ID NO.: 107)MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY Peptide-2(SEQ ID NO.: 108) DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK

FIG. 35 shows a summary of relative expression levels of prototypeCyclin B1 vaccines secreted from transfected mammalian 293F cells. Theflexible linker sequences facilitate secretion.

C1189 rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1(bold)-hCyclinB1-Peptide-2(italics)-Peptide-1 (bold-italics)-f4 (bold)][AS linkers—underlined]

(SEQ ID NO.: 109) EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS QTPTNTISVTPTNNSTPTNNSNPKPNP AS DWLVQVQMKFRLLQETMYMTVSII DRFMQNNCVPKK ASMEMKILRALNFGLGRPLPLHFLRR AS

AS TNDSITVAATAPTVTPTVNATPSAAAS

Above is the sequence of the mature secreted Heavy chain for one form ofanti-CD4012E12-cyclin B1 vaccine. The AS residues are from joiningrestriction sites. The DNA coding sequence is shown below, and thisincludes the signal peptide.

(SEQ ID NO.: 110) ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTGACTGGCTAGTACAGGTTCAAATGAAATTCAGGTTGTTGCAGGAGACCATGTACATGACTGTCTCCATTATTGATCGGTTCATGCAGAATAATTGTGTGCCCAAGAAGGCTAGTATGGAAATGAAGATTCTAAGAGCTTTAAACTTTGGTCTGGGTCGGCCTCTACCTTTGCACTTCCTTCGGAGAGCATCTAAGATTGGAGAGGTTGATGTCGAGCAACATACTTTGGCCAAATACCTGATGGAACTAACTATGTTGGACTATGCTAGTACCAACGACAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGCTGA

C1143 rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1(bold)-hCyclinB1-Peptide-2(italics)-f3 (bold)] [AS linkers—underlined].

(SEQ ID NO.: 111) EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS QTPTNTISVTPTNNSTPTNNSNPKPNP AS DWLVQVQMKERLLQETMYMTVSI IDRFMQNNCVPKK ASTVTPTATATPSAIVTTITPTATTKPAS

Above is the sequence of the mature secreted Heavy chain for one form ofanti-CD4012E12-cyclin B1 vaccine. The AS residues are from joiningrestriction sites. The DNA coding sequence is shown below, and thisincludes the signal peptide.

(SEQ ID NO.: 112) ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTGACTGGCTAGTACAGGTTCAAATGAAATTCAGGTTGTTGCAGGAGACCATGTACATGACTGTCTCCATTATTGATCGGTTCATGCAGAATAATTGTGTGCCCAAGAAGGCTAGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGACCACCATCACCCCCACCGCCACCACCAAGCCCGCTAGCTGAC911 rAB-cctHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1 (bold)-hCyclinB1-Peptide-1 (italics)-f4 (bold)](SEQ ID NO.: 113) EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASMEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDYASTNGSITVAATAPTVTPTVNAT PSAAASC911 rAB-cctHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1 (bold)-hCyclinB1-Peptide-1 (italics)-f4 (bold)]nucleic acid sequence. (SEQ ID NO.: 114)ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTATGGAAATGAAGATTCTAAGAGCTTTAAACTTTGGTCTGGGTCGGCCTCTACCTTTGCACTTCCTTCGGAGAGCATCTAAGATTGGAGAGGTTGATGTCGAGCAACATACTTTGGCCAAATACCTGATGGAACTAACTATGTTGGACTATGCTAGTACCAACGGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGCTGA

D-type Cyclin Antigen. D-type cyclins are predominantly expressed in theG1 phase of the cell cycle. The expression pattern of cyclin D1 has beenextensively studied in certain cancer types including lymphoma andnon-small cell lung cancer. Approximately 30 percent of breastcarcinomas are Cyclin D1 positive. Over expression of Cyclin D1 is now awell established criterion for the diagnosis of Mantle Cell Lymphoma, amalignant, non-Hodgkin's lymphoma which is characterized by a uniquechromosomal translocation t(11;14).

Cyclin D1—Peptide 1—bold, Peptide 2—bold-underlined, Peptide-3 italics,Peptide 4—underlined.

(SEQ ID NO.: 115) MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRL QLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPP SMVAAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI Pep-1 (SEQ ID NO.: 116)MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV Pep-2 (SEQ ID NO.: 117)QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL Pep-3 (SEQ ID NO.: 118)LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDV KFISNPPSMV Pep-4(SEQ ID NO.: 119) AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI

TABLE 1 Clone-Antibody Correlation. Name Clone Isotype PAB176AB13_22.11B6.2C6 IgG1k PAB176 AB13.22.11B6.1C3 (HS440) - subclone PAB177AB13_22.11C7.1D6 IgG2b k PAB180 AB13_22.11H12.1G1 IgG1k PAB188AB13_22.12B4.2C10 IgG1k PAB1574 PAB187 AB13_22.12E12.3F3 IgG1k PAB366PAB525 PAB530 PAB594 PAB1400 PAB1700 PAB184 AB13_22.15C11.3G12 IgG1kPAB181 AB13_22.19B5.4C11 IgG2a k PAB183 AB13_22.24A3.3F1 IgG2b k PAB178AB13_22.24C9.2A6 IgG2b k PAB189 AB13_22.2G2.1A5 IgG2b k PAB194AB13_22.3C7.1G5 IgG2a k PAB1573 PAB193 AB13_22.7G10.2D5 IgG2a k PAB1572PAB182 AB13_22.8A4.3G10 IgG1k PAB1435 PAB179 AB13_22.8F6.2C7 IgG2b kPAB190 AB13_22.9A11.2A11 IgG1 lam

FIG. 34 shows the results obtained with the various antibodies using anassay that detects signaling via CD40 ligation—read out as cell death.CD40 itself can send such signals, but the intracellular domain of FASis used for comparison when expressed in CHO cells (Fas CHO v. CHO).Briefly, CHS-S cells were transfected with expression vectors for eitherhCD40Ectodomain™ fused to FAS intracellular domain, or hCD40. Thesecells proliferate normally, but signaling through CD40 ligationactivated apoptotic signals. After 48 hours, MTT is added to the cultureand reduction in dye is measured, which is directly proportional to thecontent of active mitochondria (i.e., live cells).

ELISA. The plates were coated with either CD40 ecto (human or NHP coh)then mAbs. anti-mIgG HRP or CBD doc/then CD40 ecto (coh=cohesin, NHP=nonhuman primate, HRP=horseradish peroxidase) then mAbs and then anti-mIgGHRP or Capture is anti-mIgG then Mabs then biotinylated CD40 ecto (humanor NHP coh). Cytokine production was measured as described in theexamples above.

FIG. 35 shows the binding of various constructs when the antibody hasbeen made into a fusion protein with doc and then captures. FIGS. 36 and37 compare cytokine production with our without the addition of GM-CSFand IFNα (FIG. 36 A-D), and soluble antibodies alone (FIG. 37A-D)incubated with the DCs for 24 hours. FIG. 38A-B demonstrates the effectof various concentrations of anti-CD40 antibodies of the presentinvention on direct B cell proliferation.

B cell Proliferation. B cells from PBMC of healthy donors were enrichedby B cell enrichment kit (from BD). CFSE-labeled 5×10e4 B cells werecultured in RPMI medium containing 10% FCS in the presence of 50units/ml IL-2 for 6 days. B cell proliferation was tested by measuringCFSE dilution using flow cytometry. Surprisingly, it was found thatantibodies were able to cause B cell proliferation at various dilutions,while an immunoglobulin control and an anti-CD40 antibody (data notshown) did not.

The various constructs shown herein demonstrate the that CD40 antibodies(e.g., 12E12) are capable of strong activation as variable domains when:(1) the antibody is reconfigured as a recombinant mouse v region humanIgG4 C region chimera, and (2) the activity can be retained in thecontext of (1) with H-chain-C-terminal antigen added. These variableregion-peptide fusion proteins and/or complexes enhance greatly vaccineefficacy.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. An antibody or an antigen binding fragmentthereof, that binds to CD40, comprising light chain complementaritydetermining regions 1, 2, and 3 corresponding to SEQ ID NO: 41, SEQ IDNO: 42 and SEQ ID NO: 43, respectively and heavy chain complementaritydetermining regions 1, 2, and 3 corresponding to SEQ ID NO: 44, SEQ IDNO: 45 and SEQ ID NO: 46, respectively.
 2. The antibody of claim 1,further comprising a heavy chain constant region, wherein the heavychain constant region comprises a gamma-1, gamma-2, gamma-3, or gamma-4human heavy chain constant region or a variant of the human heavy chainconstant region.
 3. The antibody of claim 1, wherein the antibodycomprises a) at least one antibody light chain variable region of SEQ IDNO:2; and b) at least one antibody heavy chain variable region of SEQ IDNO:1.
 4. The antibody of claim 1, further comprising a light chainconstant region, wherein the light chain constant region comprises alambda or a kappa human light chain constant region.
 5. The antibody ofclaim 1, wherein the binding fragment is selected from group consistingof Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, and a diabody.
 6. The antibodyof claim 1, wherein the antibody, or antigen binding fragment thereof,comprises the polypeptide sequence of SEQ ID NO:2 and the polypeptidesequence of SEQ ID NO:
 1. 7. The antibody of claim 1, wherein theantibody alone is capable of causing dendritic cells to secrete at leastone of IL-6, MIP-1a, IL-12p40 or TNFalpha without prior activation ofthe dendritic cells.
 8. The antibody of claim 1, wherein the antibody iscapable of causing dendritic cells activated with GM-CSF and Interferonalpha to secrete at least one of IL-6, MIP-1a, IP-10, IL-10 or IL-12p40.9. A composition comprising an antibody or an antigen binding fragmentthereof, in combination with a pharmaceutically acceptable carrier ordiluent, wherein the antibody is the antibody of claim
 1. 10. Arecombinant antibody or an antigen binding fragment thereof that bindsto CD40, wherein the antibody alone is capable of causing dendriticcells to secrete at least one of IL-6, MIP-1a, IL-12p40 or TNFalphawithout prior activation of the dendritic cells, wherein the antibodycomprises light chain complementarity determining regions 1, 2, and 3corresponding to SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43,respectively, and heavy chain complementarity determining regions 1, 2,and 3 corresponding to SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46,respectively.
 11. The antibody of claim 10, wherein the antibodycomprises the heavy chain variable region of SEQ ID NO: 1 and the lightchain variable region of SEQ ID NO:
 2. 12. The antibody of claim 10,wherein the antibody is produced by a hybridoma selected from anti-CD4012E12.3F3 (ATCC Accession No. PTA-9854).
 13. The antibody of claim 10,wherein the antibody is humanized.
 14. A recombinant antibody or anantigen binding fragment thereof that binds to CD40, wherein theantibody alone is capable of causing B cell proliferation of at least10% of the B cells, wherein the antibody comprises light chaincomplementarity determining regions 1, 2, and 3 corresponding to SEQ IDNO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, respectively, and heavy chaincomplementarity determining regions 1, 2, and 3 corresponding to SEQ IDNO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, respectively.
 15. The antibodyof claim 14, wherein the percentage of B cells that proliferate is atleast 15%, 20%, 25%, 28%, 30% or 35%.
 16. The antibody of claim 14,wherein the antibody comprises the heavy chain variable region of SEQ IDNO: 1 and the light chain variable region of SEQ ID NO:2.
 17. Theantibody of claim 14, wherein the antibody is produced by a hybridomaselected from anti-CD40 12E12.3F3.
 18. The antibody of claim 14, whereinthe antibody alone is capable of causing dendritic cells to secrete atleast one of IL-6, MIP-1a, IL-12p40 or TNFalpha without prior activationof the dendritic cells.
 19. The antibody of claim 14, wherein theantibody is capable of causing dendritic cells activated with GM-CSF andInterferon alpha to secrete at least one of IL-6, MIP-1a, IP-10, IL-10or IL-12p40.
 20. The antibody of claim 14, wherein the antibody ishumanized.